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70-338 - Lync 2013 Depth Support Engineer - Dump Information

Vendor : Microsoft
Exam Code : 70-338
Exam Name : Lync 2013 Depth Support Engineer
Questions and Answers : 114 Q & A
Updated On : December 6, 2017
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70-338 Questions and Answers

Based on the network trace, you need to identify the most likely cause of the call quality issues. Which setting should you modify?

  1. Geo IP
  2. MTU
  3. DSCP value
  4. STUN port

Answer: C


You support a customer whose Microsoft Lync Server 2013 Enterprise Pool is configured with Enterprise Voice and Dial-in Conferencing. You specify a Session Initiation Protocol (SIP) Uniform Resource Identifier (URI) for the dial-in conferencing access number. You discover that the SIP URI was configured incorrectly. You need to change the SIP URI. What should you do?

  1. Run the Set-CsDialInConferencingAccessNumber cmdlet.
  2. Delete the object and recreate the access number.
  3. Run the Set-CsSipResponseCodeTranslationRule cmdlet.
  4. Run the New-CsDialInConferencingDtmfConfiguration cmdlet.

Answer: B


You deploy a Microsoft Lync Server 2013 Enterprise Pool that is configured with Enterprise Voice and Dial-in Conferencing. All client computers run Windows 7 or Windows 8, and all use Lync 2013. Users report that they are unable to share Microsoft PowerPoint presentations with users who are using certain mobile devices. They also report that they are unable to scroll through a PowerPoint presentation independent of the presentation itself. You need to ensure that while they are in conferences, users are able to share PowerPoint presentations with users who are using mobile devices. You also need to ensure that users are able to scroll through PowerPoint presentations. What should you do?

  1. Install an Office Web Apps Server on a server and configure Lync Server 2013 to communicate with Office Web Apps.
  2. Install an Office Web Apps Server on a server that is running Office 2013 and configure Lync Server 2013 to communicate with Office Web Apps.
  3. Install Office 2013 for all users and instruct them to use PowerPoint 2013.
  4. Configure a new conferencing policy and enable the AllowMultiView.

Answer: A


You support a customer whose network environment includes Microsoft Lync Server 2013 Standard Edition deployed with an Edge Server that is connected to the Internet. A user reports that when he tries to share a Microsoft PowerPoint presentation in a Lync conference, external users receive the following error message: "Some sharing features are unavailable due to server connectivity issues." You ask the customer to run the Get-OfficeWebAppsFarm cmdlet on the Office Web Application Companion (WAC) server. He provides the following result:

You need to ensure that external users are able to share and view PowerPoint presentations in Lync meetings. What should you do?

  1. Install WAC on the Edge Server.
  2. Set the ExternalUrl parameter on the WAC server and publish to the Internet by using a reverse proxy.
  3. Install WAC on the Front End pool.
  4. Set the InternalUrl parameter on the WAC server and publish to the Internet by using a reverse proxy.

Answer: B


You support a customer whose company network includes Microsoft Lync Server 2013 with web conferencing deployed at a main site named Washington and a branch site named Redmond. All users have Lync 2013 and Microsoft Outlook 2013 installed. UserA and UserB are both Lync- enabled users at your customer's main site. UserA has the Global conferencing policy and the Washington meeting configuration assigned to his account. UserB has the Redmond conferencing policy

and the Global meeting configuration assigned to his account. In online meetings that UserA schedules, users can collaborate using videoconferencing. In online meetings scheduled by UserB, videoconferencing is not available. You need to determine why videoconferencing is unavailable in UserB's meetings. Which cmdlet should you run from the Lync Server Management Shell?

  1. Get-CSConferencingPolicy -Identity Redmond
  2. Get-CSMeetingConfiguration -Identity Redmond
  3. Get-CSMeetingConfiguration -Identity Global
  4. Get-CSConferencingPolicy -Identity Global

Answer: A


You support a customer whose Microsoft Lync Server 2013 environment includes:
  • A single Standard Edition server,
  • A single consolidated Edge server, and
  • A single Forefront Threat Management Gateway 2010 server that is acting as an HTTP(S) reverse proxy.
A user reports that his attempts to join an online meeting by clicking the Join Online Meeting link are unsuccessful. Your need to troubleshoot the Join Online Meeting functionality from this workstation without using the installed Lync 2013 client. You need to achieve this goal by using the least amount of administrative effort. What should you do?

  1. Open Internet Explorer, type the URL of the online meeting into the Address bar, and then append ?sl=l to the URL.
  2. Open the Lync Options menu and select Join meeting audio from: Lync.
  3. Uninstall the Lync 2013 client, open Internet Explorer, and then enter the URL of the online meeting into the Address bar.
D.Open the Lync Options menu and set the Logging in Lync option to Full.

Answer: A


You support a customer whose network environment includes Microsoft Lync Server 2013 Standard deployed with an Edge Server that is connected to the Internet. Users are able to share screens remotely during conference calls. However, when they attempt to upload a Microsoft PowerPoint presentation, the

sharing attempt fails with an error. You need to ensure that users are able to share PowerPoint presentations during Lync conference calls. What should you do?

  1. Install and configure the Office Web Apps Server Components on the Lync Pool Server.
  2. Install and configure Silverlight on the user workstations.
  3. Install and configure an Office Web Apps Server in Lync Topology.
  4. Install and configure the PowerPoint 2013 Viewer on the Lync presenter workstations.

Answer: C


You support a Microsoft Lync Server 2013 Enterprise pool deployed in a high availability configuration for Back End Servers named Backend1 and Backend2. You execute the Get-CSDatabaseMirrorState cmdlet and discover the following errors. WARNING: Cannot connect to database server "BackEnd2.contoso.com".Message: A network- related or instance-specific error occurred whileestablishing a connection to SQL Server. The server was not found or was notaccessible. Verify that the instance name is correct and that SQL Server isconfigured to allow remote connections. (provider: Named Pipes Provider, error: 40 - Could not open a connection to SQL Server)DatabaseName
: rtcabStateOnPrimary
: PrincipalStateOnMirror
: StatusUnavailableMirroringStatusOnPrimary
: rtcxdsStateOnPrimary
: PrincipalStateOnMirror
: StatusUnavailableMirroringStatusOnPrimary
You need to resolve the connectivity issue and bring up the mirror databases to the synchronized state between the Back End Servers. What should you do?

  1. From the Front End Server, create the inbound rule on the firewall.
  2. Run the Test-CsDatabase cmdlet.
  3. Run the Invoke-CsPoolFailOver cmdlet.
  4. From the mirror Back End Server, create the inbound rule on the firewall.

Answer: D


You support a customer whose network environment includes Microsoft Lync Server 2013 Enterprise, with two Front End Servers and two Back End Servers. The Back End Servers are deployed in a high availability configuration. You create a Front End pool named pool.contoso.com and implement DNS load balancing for client access to the Lync servers. Your internal DNS records have the following configuration: _gc._tcp.contoso.com SRV priority 0, weight 100, port 3268
dc1.contoso.com_ldap._tcp.contos.com SRV priority 0, weight 100, port 389
dc1.contoso.com_kerberos._tcp.contos.com SRV priority 0, weight 100, port 88
dc1.contoso.com_sip._tls.contoso.com SRV priority 0, weight 0, port 5061, pool.contoso.com FrontEndServer1 A A A A A A Your Lync 2013 clients fail to sign in automatically. The trace log of failed sign-in attempt is as follows: INFO
:: QueryDNSSrv - DNS Name[_sipinternaltls._tcp.contoso.com]ERROR :: HRESULT failed:
80072726 = HRESULT_FROM_WIN32(::ShimWSAGetLastError()) . Failed to convert string IP to SOCKADDRERROR :: ResolveHostNameUsingGetAddrInfo - getaddrinfo(pool.contoso.com) failed WARN
:: ResolveHostName - getaddrinfo failed for pool.contoso.com ERROR :: ResolveHostNameUsingDnsQuery - DnsQuery(pool.contoso.com) failed WARN
:: ResolveHostName - DNS lookup failed for pool.contoso.com ERROR :: ResolveHostName - Name resolution for pool.contoso.com failedERROR :: ResolveHostSyncResolveHostName failed ERROR :: GetDnsResults - did not get any resultERROR :: QueryDNSSrvGetDnsResults query:
_sipinternaltls._tcp.contoso.com failed ERROR :: DNS_RESOLUTION_WORKITEM::ProcessWorkItemResolveHostName failed You need to ensure that internal Lync 2013 clients can sign in automatically. You also need to ensure that internal traffic is load balanced across the two Front End Servers. What should you do?

  1. Create the following host records:PoolA192.168.1.20PoolA192.168.1.21
  2. Create the following host records: PoolA192.168.1.10PoolA192.168.1.11
  3. Create the following service record: _sip._tls.contoso.com SRVPriority 0, weight 0, port 5061, pool01.contoso.com
  4. Create the following host records: SipA
  5. Create the following service record: _sipinternal._tls.contoso.com SRVPriority 0, weight 0, port 5061, FrontEndServer1.contoso.com

Answer: B


You support Microsoft Lync Server 2013 in your company network. Your company has four buildings on a single site. A user reports that when she calls users in another building, the call quality is poor. You receive the Quality of Experience (QoE) report: Capture device: Headset MicrophoneRender device: Headset EarphoneMicrophone timestamp error: 0.02msEcho percent microphone in: 15.21%Codec: SIRENAudio FEC: FalsePacket utilization: 32701Avg. packet loss rate: 10.21%Avg. jitter: 23msAvg. round trip: 62msAvg. network MOS: 3.71Avg. network MOS degradation (jitter):0.00%Avg. sending MOS: 2.97Avg. listening MOS: 3.17Receive noise level: -56dBoV Which of the following values is outside acceptable limits?

  1. Receive noise level: -56dBoV
  2. Packet utilization: 32701
  3. Avg. round trip: 62ms
  4. Avg. packet loss rate: 10.21%
  5. Avg. jitter: 23ms

Answer: D


You support a customer who administers Microsoft Lync Server 2013 Enterprise servers in his company. The pool named lync.contoso.com is configured with the session initiation protocol (SIP) domain contoso.com. The internal split-DNS domain contoso.com contains the following records: _gc._tcpSRV priority 0, weight 100, port 3268 dc1.contoso.com_ldap._tcpSRV priority 0, weight 100, port
389 dc1.contoso.com_kerberos._tcpSRV priority 0, weight 100, port 88
dc1.contoso.com_sipinternal._tcpSRV priority 0, weight 0, port 5061,
lync.contoso.com _sip._tlsSRV priority 0, weight 0, port 5061, lync.contoso.com EnterpriseCA
Users who are running Lync Mobile on their mobile devices report that when they attempt to retrieve calendar information, they receive an error that references a Microsoft Exchange Web Services connectivity issue. You need to ensure that users are able to receive calendar information from Lync Mobile devices. What should you do?

  1. Reconfigure Dynamic Host Configuration Protocol (DHCP) option 120 to point to DC1.contoso.local.
  2. Create the following service record in DNS: _lyncdiscover._tcp.contoso.com SRV Priority 0, weight 0, port 5061, Lync.contoso.com
  3. Reconfigure Dynamic Host Configuration Protocol (DHCP) option 43 to point to EnterpriseCA.contoso.local.
  4. Create the following host record in DNS: Autodiscover.contoso.com A

Answer: D


You deploy Microsoft Lync Server 2013 Enterprise Edition and create a pool named Lync2013pool.Contoso.local. You configure the Lync admin URL to be admin.contoso.local. The Front End and Back End roles are installed on servers named FE2013 and BE2013. Your DNS server hosts the following records:
_Sipinternaltls._tcp.contoso.local SRV priority 0, weight 0, port 5061, lync2013pool.contoso.local_tcp._kerberos.contoso.local SRV proiority 0, weight 100, port 88, DC.contoso.localDC
You attempt to open the Lync Server Control Panel and you receive an error message. You need to be able to open the Lync Server Control Panel. Which two actions should you perform? (Each correct answer presents part of the solution. Choose two.)

  1. Create the following DNS A record:Meet.contoso.local A
  2. Create the following DNS A record: lync2013pool.contoso.local A

  3. Create the following DNS SRV record: _sipinternal._tcp.contoso.local SRV priority 0, weight 0, port 5061 lync2013pool.contoso.local
  4. Create the following DNS A record: lyncdiscoverinternal.contoso.localA
  5. Modify the following DNS A record: admin.contoso.local to point

Answer: B, E


You deploy Microsoft Lync Server 2013 Enterprise Edition and create a pool named Lync2013pool.Contoso.local. You configure the Lync admin URL to be admin.contoso.local. The Front End and Back End roles are installed on servers named FE2013 and BE2013. You create the following DNS records:
_Sipinternaltls._tcp.contoso.local SRV priority 0, weight 0, port 5061, lync2013pool.contoso.localDialin A A A You attempt to open the Lync Server Control Panel and you receive an error message. You need to be able to open the Lync Server Control Panel. Which two actions should you perform? (Each correct answer presents part of the solution. Choose two.)

  1. Create the following DNS A record:Meet.contoso.local A
  2. Create the following DNS A record: lync2013pool.contoso.local A
  3. Create the following DNS SRV record: _sipinternal._tcp.contoso.local SRV priority 0, weight 0, port 5061 lync2013pool.contoso.local
  4. Create the following DNS A record: lyncdiscoverinternal.contoso.localA
  5. Create the following DNS A record: admin.contoso.localA

Answer: B, E

Microsoft 70-338 Exam (Lync 2013 Depth Support Engineer) Detailed Information

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You are required to use your legal name for the benefits and exams dashboard. The Microsoft Personal Profile is associated with your Microsoft account.
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To update your legal name, contact your Microsoft Regional Service Centre.
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You can update your Microsoft Personal Profile in the Profile Centre.
To change the name that is associated with your Microsoft Certification ID (formerly, MCP ID), contact your Microsoft Regional Service Centre.
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It can take up to 48 hours for changes in your profile to appear on your Microsoft Certification transcript. Check your transcript later to verify that your email address has been updated.
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After you update your Personal Profile, a message is sent to the email address noted in your profile. For the changes to be saved, you must follow the instructions in the email message to confirm the update. Otherwise, the email address in your profile will be removed because it has not been confirmed.
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When you access the logo tool, you are required to read and accept the MCP logo guidelines to correctly promote your relationship with Microsoft and to protect the integrity of the logos. You must sign in to the benefits and exams dashboard in order to access these guidelines.
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Logos are available for download from the benefits and exams dashboard.
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The test that you passed might not qualify you to print that specific logo. If you believe this is an error, contact the Microsoft Regional Service Centre.
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Contact the Microsoft Regional Service Centre.
Microsoft account and Profile Centre
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Microsoft account (formerly Windows Live ID) enables you to simply use your email address and password to sign in. After you create your Microsoft account, you can access all Microsoft account sites and services. If you already have an MSN Hotmail, MSN Messenger or Passport account, it is your Microsoft account.
Learn more about Microsoft account.
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If you cannot remember the Microsoft account that you used to access the benefits and exams dashboard, contact your Microsoft Regional Service Centre for assistance.
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If your Microsoft account is unused for one year, your Microsoft account may expire, and you may need to re-establish a Microsoft account.
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Log in to the benefits and exams dashboard and, under the Account menu in the top right corner of the page, click Profile settings.
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Update the following information to ensure that you receive communications from Microsoft:
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You are under no obligation to receive email messages from Microsoft. However, if you indicate that you do not want to receive communications from Microsoft through email, you cannot receive e-newsletters, such as the MCP Flash and MCT Flash. These communications provide up-to-date information on changes in training and certification resources, such as discontinued exams, Microsoft Certified Professional (MCP) benefits and special offers.
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Visit the Microsoft Profile Centre to indicate that you want to receive email messages from Microsoft.
Sign in to the Microsoft Profile Center to view and update your profile
Profile Center screenshot
Click My Contact Preferences, located on either the Profile Centre home page (shown above) or on the left-hand side of the page (shown below). Select E-Mail Address, and then click Save.
Note You can always unsubscribe from selected e-newsletters. However, if you do not select the E-Mail Address option, Microsoft cannot send you any information by email, including product notifications and news.
My Contact Preferences screenshot
Sign-up for e-newsletters: Select Manage Subscriptions on the left-hand side of the page. Tick all of the subscriptions that you want to receive, and then click Subscribe. You can also unsubscribe from publications.
Accessing your transcript
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Q. Where can I find my transcript or see which exams I have passed?
Access your transcript from the benefits and exams dashboard.
Under the section called Transcript, click on the View link.
Q. How do I give my employer or others access to my transcript via a secure connection that they can trust?
Microsoft offers a tool called Transcript Sharing, which can be accessed from the benefits and exams dashboard. Under the section called Transcript, click the Share link.
You will be asked to create an Access Code (which can be changed at any time) and given a Transcript ID. You will then provide these two codes to your selected audience, along with a URL to view your transcript.
Q. I have certifications from multiple providers; can I combine all my credentials into one transcript?
Microsoft has worked with the IT Certification Council (ITCC), along with other industry certification providers, to provide MCPs with the opportunity to create one transcript across providers. Under the Transcript section of the benefits and exams dashboard, click Create a multi-vendor transcript. You will then be asked to provide your transcript sharing codes. The transcript sharing code can be found by clicking Share your transcript.
Q. What if I cannot print or access my transcript?
If you cannot print or access your transcript, contact your Microsoft Regional Service Centre.
To ensure a prompt response:
Use the email address associated with your Microsoft Certification ID (MC ID), if possible.
Have your Microsoft Certification ID number available.
If you do not know your Microsoft Certification ID, have other information available, such as your address, telephone number, exam numbers and completion dates.
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It can take up to two weeks for Microsoft to receive and process your exam records. If it has been more than two weeks since you took your exam, and the results still do not appear on your transcript, or if you notice any other problems, contact your Microsoft Regional Service Centre.
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123inkjets is ranked 2,780,946 in the United States. 'Printer Ink Cartridges | Ink Toner Cartridges.'

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Monthly pages viewed 52,391 Monthly visits 4,200 Value per visitor $11.95 Estimated worth $3,649.96 * External links 914 Number of pages --
*Estimated data, read disclaimer.Last Updated: 08312016
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Most visitors are from Australia.
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Category: 'PrintersSupplies'
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The programming language environment is ASP.NET. It has 2 DNS records, ns2.webcontrolcenterm, and ns1.webcontrolcenterm. It is hosted by Crystaltech Web Hosting (Arizona, Phoenix,) using Microsoft-IIS6 web server.
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PING . ( 56(84) bytes of data. 64 bytes from 123inkm.au ( icmp_req=1 ttl=116 66.2 ms 64 bytes from .123inkm.au ( icmp_req=2 ttl=116 70.4 ms 64 bytes from 123inkm.au ( icmp_req=3 ttl=116 74.3 ms --- . ping statistics --- 3 packets transmitted, 3 received, 0% packet loss, time 2003ms rtt minavgmaxmdev = 66.27570.33874.3313.303 ms rtt minavgmaxmdev = 66.27570.33874.3313.303 ms
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Quick News

NCPL offers computer classes
Natrona County Public Library offers basic computer classes to help participants navigate the computer, the Internet and basic programs like Microsoft Word.
Upcoming classes are: “Intro to Computers” at 5 p.m. Wednesday, Sept. 3; “Intro to the Internet” at 10 a.m. Thursday, Sept. 11; “Intro to PowerPoint” at 1 p.m. Monday, Sept. 15; “Intro to Genealogy” at 10 a.m. Tuesday, Sept. 16; and “Intro to Microsoft Word” at 1 p.m. Monday, Sept. 17.

Stop by the library’s Reference Desk to sign up for classes, or call 577-READ, ext. 2.
Citizenship class in need of immigrants
A free class for individuals who are within one year of being eligible to apply for citizenship will begin on Monday, Sept. 8, at Casper College.
“Anyone who knows how to speak, read, and write some English and is interested in becoming a United States citizen is urged to sign up for this free citizenship class,” said Lisa Mixer, Casper College tutor coordinator and ABEGED co-director.
The class is scheduled to run for 14 weeks, each Monday from 6:30-7:30 p.m. It is sponsored by the Casper College Adult Basic EducationGED Center.
Pre-registration is required and can be done through Sept. 12 by calling Mixer at Casper College at 268-2453.
Defensive driving courses offered
Defensive driving courses for people 55 years and older are conducted on the second Tuesday and Wednesday of each month, sponsored by the Central Wyoming Senior Citizens Center.
A course is scheduled from 2-6 p.m. on Sept. 9 and 10 at the Natrona County Senior Center, 1831 E. Fourth St. Each session is taught by AARP-trained instructors.
After successful completion, participants will receive a certificate that can qualify them for a 10 percent reduction in car insurance (liability andor collision) over two years.
The cost is $10. For reservations or more information, call Georgia at 265-4678, ext. 12.
CC Greenhouse hours announced
New fall hours for the Casper College Greenhouse, featuring a variety of plants, birds and reptiles, have been announced.
According to Evert Brown, greenhouse director and Casper College biology instructor, the facility will be open to the public from noon to 3 p.m. Monday through Friday.
Many types of plants are grown in the greenhouse, representing several different climates from desert to tropical.
ors also are likely to see several varieties of birds that live and fly within the confines of the greenhouse, and also some turtles.
The greenhouse, which is located on the west side of the Loftin Life Science Center, is free and open to the public.
Bookmobile schedule
For more information, call 577-READ, or log on to
Thursday, Sept. 4: 9:30-10 a.m., Garden Square (1950 S. Beverly); 10:15-10:45 a.m., Office Max . (441 Landmark Drive); 11-11:30 a.m., Pineview School Area II (1190 S. Forest Drive); 11:45 a.m. to 12:45 p.m., Cottonwood School (2300 Bellaire Drive); 3-3:30 p.m., Park Place (1930 E. 12th); 3:40-4:10 p.m., New Directions (LifeSteps Campus); 4:40-5:15 p.m., Evansville School.
Monday, Sept. 8: 9:30-10:10 a.m., Giggles & Wiggles (1720 S. Poplar); 10:25-10:55 a.m., Sunshine Corner (2303 E. 15th); 11:05-11:45 a.m., Sagewood School Area (1230 E. 22nd); 3:20-4 p.m., Apple Tree Learning Center (60 Magnolia); 4:15-5 p.m., Robertson Road Area (Whispering Springs & King Salmon).
Tuesday, Sept. 9: 11-11:30 a.m., Powder River School; 11:35 a.m. to noon, Powder River Post Office; 3:45-4:20 p.m., Paradise Valley I (Daffodil Street); 4:30-5 p.m., Paradise Valley II (Glendo Street).
Wednesday, Sept. 10: 10-10:30 a.m., Big Tree Area (1712 S. Oak); 10:40-11:20 a.m., Mtn. Road Christian Academy (2657 Casper Mtn. Road); 11:30 a.m. to 12:30 p.m., Willard School (129 N. Elk); 2-2:30 p.m., Prince of Peace School area (South Beverly & Eighth streets); 2:45-3:20 p.m., Sagewood School Area II (1901 E. 24th); 3:30-4:10 p.m., Leaps and Bounds Preschool (615 S. David); 4:25-5:15 p.m., Boys & Girls Club (1701 E. “K” St.).
Buffalo hunt raffle benefits CWRM
An all-inclusive guided buffalo hunt to benefit the Central Wyoming Rescue Mission has been donated by various sponsors.
The winner also will receive a Pre-64 Winchester Model 70 338 Win. Mag. rifle, 3-9 Zeiss scope, gun case, sling and ammo, as well as meat processing.
Only 250 tickets will be sold at $100 each. Tickets are available at the rescue mission at 230 N. Park St., City Service Electric, Rescued Treasures and Bar-D Signs.
Checks, written to CWRM, can be sent to PO Box 2030, Casper 82602. Be sure to write “buffalo hunt” in the memo. All proceeds will go to the mission.
For more information, call Risa or Deb at 268-4474.
Counseling association sets fall conference
The Wyoming Counseling Association has scheduled its annual fall conference for Oct. 9-11 at the Parkway Plaza in Casper.
The theme is “Wyoming Winds of Change,” and Dr. Susan McCabe will be the distinguished keynote speaker.
McCabe, a nationally known expert in psychiatric mental health nursing, will be speaking at the pre-conference on “Mind and Brain: Understanding Mood States Across the Lifespan.”
She serves as an associate professor at the Fay W. Whitney School of Nursing at the University of Wyoming and is the recipient of numerous awards, including the 2007 University of Wyoming Faculty Senate Lectureship Series Award, the 2004 Basham Faculty Fellowship Award and many more.

Mental health professionals may earn up to 15 hours of continuing education at the conference. The early-bird registration deadline is Sept. 14, and payment is by check or agency voucher.
For more information about the conference, call Becky Gurtler at 265-7545, or go to
Kids’ Grief Support Group returns
After a break for the summer, the monthly meetings of the Kids’ Grief Support Group will begin again Saturday, Sept. 13.
The group will meet from 11 a.m. to 1 p.m. at Central Wyoming Hospice, 319 S. Wilson, for water balloon volleyball and pizza. They also will make plans for upcoming meetings.
The Kids’ Grief Support Group meets the second Saturday of the month throughout the school year. Kids age 6-16 who have experienced the death of someone important in their lives are encouraged to attend. Parents also are welcome.
For more information or to RSVP, call Dama at 577-4832.
Family Fun Fest raffle tickets available
Central Wyoming Hospice & Transitions will raffle numerous quilts and gift baskets at its Fourth Annual Family Fun Fest, Saturday, Sept. 20, from 11 a.m. to 3 p.m. at the Central Wyoming Fairgrounds Industrial Building.
Each year, area quilters donate their handiwork for this event. Also, area businesses pitch in by filling gift baskets with family-friendly gifts.
Tickets for quilts and for baskets are sold separately. The cost is $3 each or two for $5. The tickets are available at CWHTP, 319 S. Wilson, and at Kalico Kat Quilt Shop, 350 W. Collins, also will be available at the event.

The Family Fun Fest is Casper’s largest indoor picnic and will feature games for all ages and prizes.
For more information, call Denise at 577-4832.
Book drive under way
Joan Anderson of Casper is conducting a book drive for the Natrona County Detention Center.
Anderson is asking for donations of paperbacks, in decent condition, of all genres: classics, action, Bibles, mysteries, romance, everything.
She noted that books in Spanish especially are needed.
Anderson plans to donate books to the detention center in September. Anyone with donations is asked to call Anderson at 472-3720.
Platte River Revival announced
The Second Annual Platte River Revival will be held on Saturday, Sept. 20, from 9 a.m. to noon, with check-in at Mike Lansing Field.
Residents are encouraged to start forming a team, perhaps with colleagues or neighbors.
“Volunteers will help remove debris and Russian olive branches, and plant trees in designated locations along the North Platte River,” said Jolene Martinez, Keep Casper Beautiful director.
In preparation for volunteers, Bureau of Land Management fire crews began cutting Russian olives, a non-native, invasive species, recently in what is expected to take a few weeks to complete.
Last year, volunteers removed 381,380 pounds of debris from the river and its banks, including appliances, tires and vehicles in the area just east of Bryan Stock Trail to the White Water Park.
Eleven native species trees were planted in Riverview Park.
“This year, the Revival’s operations committee is looking at areas along the river from Casper’s east city limit to just past Morad Park,” Martinez said.
A barbecue will follow, and there will be a group picture of all volunteers.
For more information or to sign up to volunteer, visit the Web site link at
The Platte River Revival is a Keep Casper Beautiful event in conjunction with National Public Lands Day and is organized by a partnership of public and private organizations.
Bereavement support groups in Glenrock
North Platte Home Health and Hospice are conducting Bereavement Support Group meetings in Glenrock to provide support, discussion and conversation for those who have lost a loved one.
Meetings are held the third Friday of the month at the Glenrock Senior Center, 615 W. Deer. Led by Chaplain Gayle Unruh, the meetings are held from noon to 1:30 p.m.
Lunch is provided, and the public is invited to attend these free meetings.
Hospice is a health care program that provides physical, emotional and spiritual support with life-limiting illnesses to enable the best quality of life for the patient.
It provides educational and emotional support to the patient's family to make their remaining time with the patient as meaningful as possible. Additionally, hospice provides grief support to family members following the death of the patient.
North Platte Home Health and Hospice is owned by Amedisys ., a provider of home health and hospice services at more than 480 sites across the United States and Puerto Rico.

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Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection

Human adenovirus (HAdV) infection constitutes a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). The incidence of HAdV infection ranges from 5 to 30 %, with pediatric recipients showing the highest rates of infection with up to 83 % lethality [1–6]. Monitoring for HAdV infection and therapeutic intervention (reduction of immunosuppression, antiviral treatment) may reduce mortality due to HAdV in pediatric HSCT recipients [7]. However, antiviral treatments for HAdV infection with agents like cidofovir and ribavirin are associated with toxicity and may result in delayed immune reconstitution. Previous studies clearly indicate that T cells, the most potent effectors of the human immune system, are crucial for HAdV clearance [2]. It was demonstrated that children with HAdV-associated mortality had no HAdV-specific T cells, whereas patients who cleared HAdV infection showed HAdV-specific T-cell responses [2, 8]. Adoptive transfer of HAdV-specific T cells offers an effective and non-toxic immunotherapeutic strategy to reduce or prevent the clinical manifestation of HAdV in HSCT recipients with no or low numbers of HAdV-specific T cells [2, 8–12]. Monitoring HAdV-specific T-cell immunity may improve risk assessment in HSCT recipients and enhance treatment efficacy by determining the optimal time point for adoptive T-cell transfer. The median time between the first detection of HAdV DNA in the blood and the onset of symptoms is 3 weeks, which therefore seems to be the optimal time point for adoptive T-cell transfer [2, 13, 14]. Since the generation of short-term in vitro generated virus-specific T-cell lines takes about 3 weeks including quality controls, the production should start even earlier at the time of high viral load in stool (>106 copies) [12, 15].
The 70 different human HAdV types identified to date are divided into seven species (A to G) [16, 17]. Type 31 (of species HAdV-A) and HAdV 1, 2, and 5 (of species HAdV-C) are the most prevalent types in HSCT recipients [4–7]. Occasionally, types of species HAdV-B can be observed in adult HSCT recipients [18]. The major capsid protein hexon serves as an immunodominant target antigen across the different HAdV types, but few hexon-derived epitopes have been identified as immunodominant so far [13, 19–23]. Most of these epitopes are highly conserved, demonstrating that HAdV-specific T cells can cross-react across HAdV species and may therefore provide protection against a wide range of HAdV types [20]. HAdV-specific T-cell responses to the recombinant hexon protein, the overlapping peptide pool covering the complete hexon sequence, HLA-restricted peptides, and whole viral lysates have been investigated. A study by Feuchtinger et al. revealed that 10.5 % of donors had a specific T-cell response to the whole adenovirus but no response to the hexon protein, while 17 % of donors had no detectable T-cell response to HAdV [11]. Moreover, Zandvliet et al. detected specific CD8+ T cells in 616 healthy donors (37.5 %) after stimulation with the 15-mer hexon peptide pool, but only 316 donors (18.8 %) had specific T cells for known CD8+ hexon epitopes [24]. Sukdolak et al. observed a specific T-cell response to the 15-mer hexon peptide pool in 73 % of HAdV seropositive healthy donors, while 30 % were classified as high responders and 43 % as low responders [25]. Interestingly, 27 % of all HAdV seropositive healthy donors tested showed no response to the hexon peptide pool. These results underline the need to identify more immunogenic T-cell epitopes to improve the selection of HAdV-specific T cells for adoptive transfer and the immunomonitoring of high-risk patients.
T-cell epitopes can be identified by direct or reverse immunology. Various computer algorithms have been developed over the past years that allow for the prediction of peptide binding to MHC class I and II molecules, proteasome cleavage patterns and transporter associated with antigen processing translocation [26]. Naturally presented CD8+ T-cell epitopes are usually among the top-scoring 2 in 80 % of all predictions, whereas the reliability of CD4+ T-cell epitope prediction is much lower due to the more variable pocket binding behavior of MHC class II molecules [27]. SYFPEITHI [26, 28, 29], BIMAS [26, 30] and NetChop [31] are the most widely used algorithms to identify cytotoxic T lymphocyte (CTL) epitopes in viral, microbial, and tumor antigens. These well-established algorithms, which have been validated and compared [26], were employed in this study to predict new HAdV epitopes.
The major focus of this study was to identify and evaluate novel immunodominant HAdV-specific T-cell epitopes by analyzing the main structural proteins, hexon and penton. HLA-A*01-, A*02-, A*03- and B*08-restricted peptide epitopes within conserved protein regions (Table 1) were pre-selected based on the predictions of several established computer algorithms. Immunogenicity of the top-ranked epitopes was investigated by established methods: IFN-γ-based EliSpot, cytokine secretion assay (CSA), peptide MHC (pMHC) multimer staining and multicolor flow cytometry. Four of the selected peptide candidates were classified as low immunodominant and two as high immunodominant according to the number of responders in the healthy donors and HAdV-infected HSCT recipients. This paper describes for the first time the immunogenic potential of penton-derived epitopes and demonstrates that the penton, as an immunological target, it is not secondary to the hexon. Expanding the repertoire of immunodominant HAdV-specific T-cell epitopes will enable more precise immunomonitoring and more effective multi-epitope-based T-cell therapy by targeting epitopes presented in a broader array of HLA molecules. Table 1
Predicted peptide candidates used for HAdV-specific T-cell screening in healthy donors

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Award-Winning Chef Elizabeth Falkner Reveals Her Struggle with Atopic Dermatitis to Highlight the Physical and Psychological Impact of the Disease

CAMBRIDGE, Mass. and TARRYTOWN, N.Y., July 12, 2016 PRNewswire -- Celebrity chef, restaurateur, and media personality Elizabeth Falkner has teamed up with Sanofi Genzyme, Regeneron Pharmaceuticals, ., the National Eczema Association, and the Dermatology Nurses Association to launch Understand AD, a national awareness campaign focused on educating people about moderate-to-severe atopic dermatitis (AD), a potentially serious, chronic inflammatory skin disease.1 Falkner is speaking out for the first time about her own struggle with the disease to drive awareness about the physical impact and effects on quality of life for people living with atopic dermatitis, and to encourage others to speak up about their experience.
"I have been living with the challenges of atopic dermatitis for more than 20 years. At its worst, my atopic dermatitis causes constant, unbearable itching, scabbing, visible rashes on my body and even bleeding, and that's only the physical part," says Elizabeth Falkner. "Having atopic dermatitis can affect many aspects of a person's life – physically and emotionally – and yet many people don't understand the severity and impact. I joined Understand AD to empower people to have more open conversations with their doctors and loved ones about the impact this disease has on their lives."
Atopic dermatitis is a chronic, systemic inflammatory disease characterized by rashes and can include intense itching, skin dryness, cracking, redness, crusting and oozing.1,2,3 Though symptoms can appear on the surface of the skin all over the body,4 advances in research have provided new insights on the cause of atopic dermatitis.5 Scientists now believe AD is caused in part by systemic allergic inflammation that results from a malfunctioning immune system.4,6 The physical symptoms are challenging and impact people's sleep and daily lives and the disease can also make people feel self-conscious and embarrassed about their appearance.7,8,9
"Understand AD aligns with our mission to educate the public and support patients impacted by atopic dermatitis," says Julie Block, President and CEO, National Eczema Association. "Unfortunately, there's a misperception that atopic dermatitis is just a 'skin condition' that people can deal with on their own, but in reality, it's an immunological disease that has a huge impact on patients' lives. We want people living with this disease to know that they're not alone and that we're committed to advocating for better care and treatments, providing support and raising the level of awareness about this serious, and often overlooked, disease."
An estimated 1.6 million adults in the United States live with uncontrolled moderate-to-severe atopic dermatitis.10 Researchers continue to discover more about atopic dermatitis and there is still a need for additional treatment options for atopic dermatitis.
"Our community of nurses on the front lines see people every day who are suffering with atopic dermatitis," says Donna Beyer, MSN, RN, DNC, President of the Dermatology Nurses Association. "But there is still a gap in public awareness about this disease and a clear need for continued education and supportive resources for patients. We're excited to join Understand AD to help educate about the disease and to drive the dialogue that atopic dermatitis is more than skin deep."
.UnderstandADm to learn more about moderate-to-severe atopic dermatitis, get connected with advocates such as the National Eczema Association and Dermatology Nurses Association, and hear from award-winning chef, media personality and restaurateur Elizabeth Falkner who has lived with atopic dermatitis for the past 20 years.
About SanofiSanofi, a global healthcare leader, discovers, develops and distributes therapeutic solutions focused on patients' needs. Sanofi is organized into five global business units: Diabetes and Cardiovascular, General Medicines and Emerging Markets, Sanofi Genzyme, Sanofi Pasteur and Merial.
Sanofi Genzyme focuses on developing specialty treatments for debilitating diseases that are often difficult to diagnose and treat, providing hope to patients and their families.
Genzyme® is a registered trademark of Genzyme Corporation. Sanofi® is a registered trademark of Sanofi. .

About Regeneron Pharmaceuticals, .Regeneron is a leading science-based biopharmaceutical company based in Tarrytown, New York that discovers, invents, develops, manufactures, and commercializes medicines for the treatment of serious medical conditions. Regeneron commercializes medicines for eye diseases, high LDL cholesterol and a rare inflammatory condition and has product candidates in development in other areas of high unmet medical need, including rheumatoid arthritis, asthma, atopic dermatitis, pain, cancer, and infectious diseases. For additional information about the company, please visit .regeneronm  or follow Regeneron on Twitter.
About the National Eczema AssociationThe National Eczema Association (NEA) is a non-profit 501 (3) patient advocacy organization whose mission is to improve the health and quality of life for individuals with eczema through research, support, and education. In the United States alone, over 10% of the population has some form of atopic dermatitiseczema. NEA was founded in 1988 by a group of patients, medical professionals, and parents to help individuals and families living with this skin disease live healthier lives. Through a variety of educational materials, including a quarterly patient-oriented magazine, a monthly electronic newsletter, and trustworthy website, the NEA reaches out to a diverse audience that includes eczema patients, caregivers, medical professionals, and other stakeholders. NEA also conducts patient conferences and participates in a wide-variety of medical symposiums. NEA is active year round to promote eczema awareness, break through stereotypes and address issues critical to patient care. Advocacy efforts include advancing increases in skin disease research funding through the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health, as well as increasing public understanding regarding the burden of eczema. NEA provides a network of support groups, an up-to-date website with the latest research and treatment information, a Seal of Acceptance program for over-the-counter products to help eczema patients navigate the myriad of products necessary for their daily skin care regimen, and a research program to advance scientific knowledge and care.  All NEA programs and services result in benefits for eczema patients and their families. NEA does not endorse specific products. For more information about the National Eczema Association, visit .nationaleczema, contact at infonationaleczema, or call 1-800-818-7546.
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NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells

Abstract The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFβ). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1Snail1, SNAI2Slug and ZEB2Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer “stem-like” phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-κB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-κB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-κB activity. Furthermore, inhibition of NF-κB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-κB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.

Citation: Kumar M, Allison DF, Baranova NN, Wamsley JJ, Katz AJ, Bekiranov S, et al. (2013) NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells. PLoS ONE 8(7): e68597. doi:10.1371journal.pone.0068597
Editor: Srikumar P. Chellappan, H. Lee Moffitt Cancer Center & Research Institute, United States of America
Received: January 14, 2013; Accepted: May 30, 2013; July 30, 2013
: 2013 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work was supported by National Institutes of Health grants R01CA132580, R01CA104397 (to M.W.M.), and R01CA136705 (to D.R.J.). All funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cancer development from early pre-malignant neoplasm to full metastatic disease is a multistep process that involves tumor epithelial-stromal interactions, angiogenesis, and infiltration of tumor-associated pro-inflammatory cells [1], [2]. An emerging hypothesis proposes that this milieu of cell-cell interactions, growth factors, and cytokines known as the tumor microenvironment, stimulates morphogenesis within tumor cells referred to as the epithelial-to-mesenchymal transition (EMT) [3]–[5]. EMT induces a redistribution of intracellular architecture, decreased cell-cell adhesion, and loss of cellular polarization. Carcinoma cells that have undergone EMT are characteristically motile, invasive and highly metastatic. Over the past several years, EMT has also been recognized as a de-differentiation program attributed to generation of tumor-initiating or cancer-initiating cells (CICs) that are important in the maintenance of cancer “stemness” [6]–[9].

Although multiple cytokines and growth factors induce EMT, one of the best studied factors is transforming growth factor beta (TGFβ) [2], [3], [10]–[13]. Stimulation of cells with TGFβ results in expression of the EMT master-switch transcription factors, TWIST1, SNAI1Snail, SNAI2Slug, and ZEB2Sip1 that together differentially regulate genes to promote the mesenchymal phenotype [10], [12]. While extensive research details the ability for TGFβ to induce EMT, evidence indicates that tumor necrosis factor (TNF) further potentiates the transition [14], [15]. During cancer progression, secretion of TGFβ within the tumor microenvironment occurs through many different cell types, including tumor-associated fibroblasts, while secretion of TNF originates from tumor-associated M2 macrophages [3], [16], [17]. A prevailing hypothesis in the field is that exposure of cancer cells to these cytokines within the tumor microenvironment promotes EMT, de-differentiation, and the formation of CICs [2], [5], [17].

TNF is a powerful pro-inflammatory cytokine that stimulates signaling cascades to activate nuclear factor kappa B (NF-κB). As a transcription factor, NF-κB plays a key role in the expression of genes involved in cancer initiation and progression. Upregulation of NF-κB activity often occurs in primary solid and hematological tumors, directly correlating with de-differentiated morphology, advanced tumor stage, and poor clinical prognosis [18]. Importantly, NF-κB has been linked to mammary CICs [19], [20]. NF-κB induces and maintains EMT in model systems through two mechanisms, upregulation of EMT master-switch transcription factors [21]–[24] and stabilization of Snail [25]. NF-κB is composed of five Rel family members: RelAp65, RelB, cRel, p50 and p52. In unstimulated cells, inhibitory IκB subunits associate with NF-κB dimers and sequester them in the cytoplasm. Upon cellular stimulation by pro-inflammatory cytokines, IκBα is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated by the SCF-type E3 ligase, E3RSIκBβ-TrCP and degraded by the 26S proteasome [26]. Liberated NF-κB then translocates to the nucleus to activate gene expression by recruiting transcriptional coactivators [27]. Our laboratory has shown that posttranslational modifications on RelA are required for full NF-κB transcriptional activity [27]–[30].

Although EMT in breast cancer models requires NF-κB activity [31], the role of this transcription factor in stimulating EMT and developing CICs in NCSLC has not been thoroughly examined. However, strong evidence exists for the presence of NSCLC stemprogenitor cells in primary adenocarcinomas and established cell lines [32]–[35]. Here, we demonstrate that coordinated activation of TNF and TGFβ signaling cascades effectively induces EMT and the expression of genes related to de-differentiation and stemness. Further, we show that mesenchymal NSCLC cells possess constitutively active NF-κB, and that inhibition of NF-κB decreases EMT, CIC formation, and metastatic potential.
Materials and Methods Cell culture and reagents
NSCLC lines A549, H359, H1299, and H157 were obtained from ATCC and maintained as 2D cultures in DMEM (CellGro), 10% FBS (Invitrogen) and penicillinstreptomycin (Invitrogen). The antibodies used include: E-cadherin (BD Pharmingen- 610404), N-cadherin (BD Pharmingen- 610920), Vimentin (V6630), Fibronectin (BD Pharmingen- 610078), α-Tubulin (Sigma T6793), HMGA2 (Biocheck 59170AP), Twist1 (Cell Signaling 4119), Snail1 (Cell Signaling 4719), Sip1 (SCBT sc-48789), Slug (Abcam ab27568), IκBα (pS32, Cell Signaling 2859), IκBα (SCBT sc-371), RelA (pS536, Cell Signaling 3031), RelA (SCBT sc-372), and M2-Flag (Sigma F1804). Baculogold protease inhibitors were obtained from BD Biosciences. TGFβ (PHG 9204) and TNF (PHG 3015) were purchased from InvitrogenLife technologies. All other chemicals were from Sigma.
Three-dimensional multicellular spheroid cultures
Three-dimensional multicellular spheroid cultures were created using a modified hanging droplet method [36]. Cells were grown to approximately 80% confluence on standard tissue-culture plates. The cells were subsequently trypsinized, resuspended in DMEM10% FBS, and counted. To create 25,000 cell spheroids, the cell suspension was diluted to a concentration of 1,000,000 cellsml, and 25 µl of the cell suspension were pipetted onto the underside of a sterile 10 cm tissue-culture plate lid. Each lid holds approximately fifty droplets. After loading the droplets, the lid was placed onto a tissue culture plate containing 6 mL of sterile PBS and incubated for 48 hours to facilitate cellular aggregation and spheroid formation. The freshly formed spheroids were then transferred into 10 cm suspension plates containing DMEM and 2% FBS to prevent cell attachment to the dish. Suspension plates were made by adding 8 ml of poly-HEMA solution (Sigma-Aldrich P3932, 10 mgml) in 95% ethanol to sterile polystyrene petri dish plates (Fisher Scientific). The plates were then incubated for 24 hours in a sterile environment to allow the ethanol to evaporate. Prior to use, plates were washed with sterile PBS to remove any residual ethanol or other contaminants. Each suspension plate holds up to 100 spheroids. After transfer, the spheroids were treated with vehicle or with 10 ngml TNF and 2 ngml TGFβ, and incubated for 48 hours. After incubation, cells were subjected to a second treatment of vehicle or TNF and TGFβ, and incubated an additional 48 hours. The spheroids were then collected and analyzed by various assays.
Immunofluorescence Microscopy
A549 cells were seeded on glass coverslips and subjected to EMT induction or left untreated. After induction, the cells were fixed in 100% methanol and subsequently incubated with primary antibodies to the extracellular domain of E-Cadherin (SCBT, sc-7870). An AlexaFlour-conjugated, goat anti-rabbit antibody (Invitrogen) was used as a secondary antibody, and indirect immunofluorescence of E-Cadherin was imaged using a Nikon E3800 fluorescence microscope.
Migration and Invasion
In vitro migration and invasion assays were carried out according to the manufacturer's protocol (BD Biosciences). 2D and 3D cultures were disaggregated by trypsin and subsequently counted. 1×105 cells (migration) or 1×104 cells (invasion) were seeded in plain DMEM in the top well of a transwell control plate (BD 354578) or Matrigel invasion plate (BD 354480). The bottom well was loaded with DMEM containing 10% FBS as a chemoattractant, and the plates were incubated for eight hours (migration) or twenty-four hours (invasion) at 37°C and 5% CO2. Afterwards, cells on the upper side of the membrane were removed, and the remaining cells were fixed in 100% methanol and stained with 0.1% crystal violet. The stained cells were imaged and quantified using Adobe® Photoshop.
Tumor model
Monolayer (2D) and 3D A549 cultures that had been left untreated or treated with TNF and TGFβ were trypsinized, resuspended in DMEM0.5% FBS, and carefully counted and diluted in the appropriate volume for injection. Cells were subcutaneously (SC) injected into female outbred Crl:NUNU nude mice (Charles River). Five mice were injected per experimental condition. All animal studies were performed as three independent experiments. Mice were sacrificed forty days post-injection. The primary SC tumors were removed and weighed. Additionally, the lungs were removed, fixed in formalin, and surface lung metastases were counted. To quantify the amount of total tumor burden in the formalin fixed lung tissue, genomic DNA was extracted [37] and assayed for the presence of human genomic material as described using quantitative real time-polymerase chain reaction (QRT-PCR) primers specific to human endogenous retrovirus-3 (ERV3, Table S1) [38], [39].

This study was carried out in strict accordance with recommendation from the Animal Care and Use Committee (ACUC) of the University of Virginia. The protocol was approved by ACUC Number 3914. All experiments were terminated after 40 days at which time SC tumors were less than 1.0 cm3 in size; thus, restricting tumor burden. All efforts were made to minimize pain and suffering.
QRT-PCR, Immunoblots, and Electrophoretic mobility shift assays (EMSAs)
QRT-PCR and immunoblot experiments were carried out as previously described [28]. PCR primers are shown in Table S1. Nuclear extracts were prepared using spheroids from A549.V and A549.I cell lines treated with or without TNF and TGFβ. EMSAs and supershift assays were performed as described previously [40].
Where appropriate, comparisons between experimental groups were carried out by performing a one-tailed Student's t test in Microsoft excel. Data for all experiments was considered statistically significant when p<0.05.
Results A model to study EMT in NSCLC
TNF has been shown to potentiate TGFβ-mediated EMT through the activation of co-stimulatory pathways [15]. To confirm this observation in our three-dimensional (3D) model, a timecourse was performed using both cytokines in tandem and alone. Multicellular spheroid cultures were created using a modified hanging droplet method [36]. After two days, spheroids were suspended in poly-HEMA coated plates and treated every two days with the indicated cytokines to induce EMT (Figure 1A). Samples were collected from untreated (0 days) and cytokine-treated cultures (1–8 days). Epithelial (E-cadherin) and mesenchymal (N-cadherin, Vimentin, and Fibronectin) markers were measured by immunoblot. Treatment with TNF resulted in a modest increase in N-cadherin and Fibronectin, but failed to show differences in other markers (Figure 1B). Consistent with the induction of EMT, TGFβ treatment resulted in a loss of E-cadherin expression and an increase in N-cadherin, Vimentin, and Fibronectin. Moreover, co-stimulation with TNF and TGFβ yielded a more mesenchymal phenotype and persisted throughout the eight day time course (Figure 1B). Importantly, stimulation with TNF and TGFβ effectively induced EMT in both A549 and H358 cell lines within four days of treatment, compared to H1299, which already shows changes in E-cadherin and vimentin (Figure 1C). Based on results in Figure 1, we used the four day timeframe throughout our remaining experiments.

Figure 1. Establishment of three-dimensional multicellular culture model for EMT studies.

(A) A timeline illustrates the procedure used to create a three-dimensional mesenchymal cell population from confluent monolayers. (B) Spheroid cultures of A549 cells were treated, with TNF, TGFβ, or both cytokines every forty-eight hours for the indicated times. Immunoblot analysis measured changes in epithelial (E-cadherin) and mesenchymal (N-cadherin, Vimentin, and fibronectin) markers over an eight day timecourse. (C) 3D cultures of multiple NSCLC cell lines (A549, H358, H1299) were incubated for ninety-six hours in the absence or presence of TNF and TGFβ. Epithelial and mesenchymal markers were subsequently measured by immunoblot. Results from Figure 1B and 1C are representative examples from at least three independent experiments; α-tubulin acts as a protein loading control.
3D cultures undergo EMT more efficiently than 2D cultures
To determine whether 3D A549 cultures undergo EMT more efficiently than two-dimensional (2D) monolayer cultures, we measured expression of epithelial and mesenchymal markers in response to stimulation with TNF and TGFβ as described in Figure 1. Following cytokine treatment, 3D cultures show significant loss of CDH1E-cadherin expression when compared to 2D cultures (Figure 2A). Moreover, the spheroids also possess increased expression of mesenchymal markers VIM, HMGA2, and the EMT master-switch transcription factors, TWIST1, SNAI1Snail1, SNAI2Slug and ZEB2Sip1 (Figures 2A and 2B). Immunoblot analysis of spheroid cultures confirm that the differential mRNA expression resulted in a corresponding change in protein levels (Figure 2C). Additionally, we examined changes in cellular morphology and E-cadherin localization by microscopy. Both 2D and 3D cultures were treated with cytokines as described, trypsinized, re-plated on glass coverslips, and indirect immunofluorescent staining was carried out eighteen hours later. As expected, untreated monolayer and spheroid A549 samples showed robust E-cadherin expression, though the junctional localization appeared diminished in cells from the 3D cultures (Figure S1). Furthermore, cells derived from cytokine-treated spheroids displayed enhanced loss of E-cadherin when compared to 2D treated samples, suggesting that 3D cultures underwent more efficient EMT. Results shown in Figure 2 and Figure S1 illustrate significant EMT induction in 3D cultures as measured by changes in mesenchymal markers, EMT master-switch transcription factor expression, and cellular morphology.

Figure 2. Three-dimensional cultures show enhanced sensitivity to cytokine treatment.

(A and B) Monolayer (2D) and 3D cultures of A549 cells were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. Expression of epithelial markers (CDH1), mesenchymal markers (VIM, HMGA2), and EMT master-switch transcription factors (TWIST1, SNAI1, ZEB2, SNAI2) were measured by QRT-PCR. (C) Immunoblot analysis of 3D A549 cultures, left alone (No Add) or treated with TNF and TGFβ (TNFTGF), was performed on E-cadherin, Vimentin, HMGA2, Twist1, Snail1, Sip1, Slug, and α-tubulin. Results in Figure 2A and 2B were normalized to GAPDH, and are calculated mean ± S.D, *p<0.05, N = 3. Immunoblots in Figure 2C are representative example from at least three independent experiments.
Mesenchymal NSCLC cells are invasive and endogenously express genes known to promote stem-like properties
Phenotypically, mesenchymal cells have high migration rates and secrete enzymes that degrade extracellular matrix to facilitate cellular invasion. Using in vitro transwell assays, we measured the migration and invasion characteristics of A549 cells grown as either 2D or 3D cultures. Interestingly, untreated 3D spheroid cultures showed higher migration rates than 2D monolayer cultures (Figure 3A, left). However, treatment of 3D cultures with TNF and TGFβ further potentiated migration when compared to untreated 3D cultures. Spheroids treated with cytokines invaded through Matrigel more effectively than any other condition (Figure 3A, right). Additionally, cytokine treated A549 spheroids displayed upregulated expression of MMP9, LOX, and COL22A1 (Figure 3B), genes known to potentiate invasion [41], [42]. These results demonstrate that culturing 3D spheroids in the presence of TNF and TGFβ establishes a highly invasive mesenchymal population. Finally, cytokine-treated spheroids showed endogenous upregulation of markers associated with de-differentiation and maintenance of CICs [43]–[48], including KLF4, SOX2, POU5F1Oct4, MYCN, and KIT (Figure 3C). Data shown in Figure 3 indicate that co-stimulation of spheroids with TNF and TGFβ promotes phenotypic changes in A549 cells that result in increased invasion and expression of gene products associated with stem-like properties.

Figure 3. Efficient induction of EMT promotes invasion and the expression of genes required to maintain CICs.

Monolayer and 3D A549 cultures were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. (A) Cells were disaggregated and subsequently subjected to migration and invasion assays. (B and C) Expression of invasion (MMP-9, LOX, COL22A1) and stem cell markers (KLF4, Sox2, POU5F1, MYCN, and KIT) was measured by QRT-PCR. Results in Figure 3 are calculated mean ± S.D, *p<0.05, N = 3. Results from 3B and 3C were normalized to GAPDH.
Mesenchymal cells are highly metastatic and display cancer initiating phenotypes
To examine whether induction of EMT promotes the development of CICs in vivo, we utilized a xenograft tumor model in nude mice. TNF and TGFβ treated 2D and 3D cultures were disaggregated and cell suspensions were SC injected into the right flank of nude mice. Forty days later, animals were sacrificed and SC tumors were resected and weighed while the lungs were excised and scored for surface metastases. To our surprise, TNF and TGFβ-treated cells did not form SC tumors to the same extent as cytokine-treated 2D cultures (Figure 4A, left). However, examination of the lung surface in these mice revealed extensive metastasis (Figure 4A, right). The only plausible explanation for these results is that mesenchymal cells from 3D cultures invaded and metastasized to the lung without developing SC tumors.

Figure 4. Cytokine-treated 3D cultures contain CICs with increased metastatic potential.

(A) Monolayer and 3D A549 cultures were treated with TNF and TGFβ for ninety-six hours. Cells were disaggregated and SC injected into nude mice (1×106 cellsanimal). Forty days later, the primary SC tumors were resected and weighed. Additionally, the lungs were excised and the number of surface metastases were determine. (B) Monolayer and 3D A549 cultures were either left untreated or treated with TNF and TGFβ and limiting cell numbers (1×103animal) were SC injected into nude mice to evaluate the presence of CICs. Metastasis was evaluated by surface lung tumor count and lung tumor burden was evaluated using genomic QRT-PCR to detect human DNA in total lung tissue. Weight and lung metastases data from Figure 4 are mean ± S.D. of five mice per condition, *p<0.05, N = 3 independent experiments. Genomic QRT-PCR data from Figure 4B are normalized to total lung tissue (mg).

Measuring the extent of metastasis under limiting cell dilution proves a reliable test for the presence of enriched CICs in epithelial-derived tumors [49]. Therefore, experiments were repeated using one-thousand cells per SC injection. Cell suspensions, derived from TNF and TGFβ treated spheroids, produced more surface lung metastases under limiting cell dilution than cytokine-treated monolayers or untreated 3D cultures (Figure 4B left). Limiting cell dilution assays indicate that induction of EMT in 3D cultures produces a CIC population that effectively metastasizes to lung. As expected, analysis of DNA isolated from mouse lungs confirmed the presence of metastatic burden and verified that the lesions were of human origin (Figure 4B right). We conclude from the experiments in Figure 4 that de-differentiation, CIC formation, and metastatic potential are all significantly enhanced in EMT-induced spheroid cultures.
NF-κB is constitutively active in 3D cultures and is required for induction of EMT
TNF, a potent NF-κB activator, enhances induction of EMT in NSCLC cell lines. Therefore, we assessed whether EMT induction results in activation of NF-κB signaling by immunoblot. Interestingly, mesenchymal A549 spheroids displayed constitutive IKK activity as measured by phospho-specific antibodies that detect IκBα pS32 and RelA pS536 (Figure 5A and Figure S2A). Change in E-cadherin and Vimentin levels confirmed efficient EMT in the cytokine-treated spheroids. Moreover, QRT-PCR experiments demonstrated increased expression of NF-κB-regulated genes IL8 and BIRC3cIAP2 in mesenchymal 3D cultures (Figure 5B). Collectively, these data indicate that cytokine-treatment of 3D A549 cultures results in the increased phosphorylation of IKK-regulated substrates and constitutive NF-κB transcriptional activation.

Figure 5. Mesenchymal cells display constitutive NF-κB activity.

Monolayer and 3D cultures of A549 cells were incubated with cytokines for ninety-six hours. (A) Mesenchymal A549 cells display constitutive NF-κB activated pathways, as determined using phospho-specific antibodies to IκBα and RelA. (B) Untreated and TNF and TGFβ stimulated 2D and 3D cultures of A549 cells were harvested and analyzed for expression of NF-κB regulated genes by QRT-PCR. (C and D) Three dimensional cultures of A549.V (vector control) and A549.I (SR-IκB) were incubated for ninety-six hours in the absence or presence of TNF and TGFβ. (C) Immunoblots confirm the expression of the Flag-tagged SR-IκBα in the A549.I line, which successfully blocked nuclear translocation and DNA binding, as measured by EMSA. (D) QRT-PCR confirmed the inability of A549.I cell to upregulate NF-κB-regulated genes following TNF and TGFβ treatment. Immunoblots in Figure 5A are a representative example from three independent experiments. Results in Figure 5B and 5D are calculated mean ± S.D, *p<0.05, N = 3. RNA values were normalized to GAPDH.

To determine the importance of NF-κB activity during induction of EMT in NSCLC cell lines, stable clonal pools expressing the super-repressor IκBα (SR-IκBα) were generated. The SR-IκBα is resistant to proteasomal degradation, and consequently sequesters NF-κB in the cytosol. Cells expressing the SR-IκBα protein therefore display an inhibition of NF-κB-mediated transcription [50]. Figure 5C (top) confirms expression of Flag-tagged SR-IκBα in A549 stable cells (A549.I) compared to empty vector control cells (A549.V). Furthermore, nuclear protein extracts from A549.I spheroid cultures, treated with TNF and TGFβ, lacked NF-κB DNA binding activity as compared to A549.V extracts (Figure 5C, bottom). Supershift experiments confirm that the NF-κB activity is composed predominantly of a RelA-p50 heterodimer complex (Figure S2B). QRT-PCR assays show repressed cytokine-mediated induction of IL8 and BIRC3cIAP2 in A549.I cells when compared to control cells A549.V (Figure 5D). In contrast to high doses of TNF (100 ngml), low doses (10 ngml) did not result in a loss of cell viability in A549.I lines, since expression of the house keeping gene, HPRT, did not change and was used for normalization in Figure 5D. These data verify that SR-IκBα expression in the A549.I cell line effectively blocks NF-κB transcriptional activity.
Characterization of NF-κB in potentiating the mesenchymal phenotype
NF-κB has been shown to regulate the expression of EMT master-switch transcription factors in multiple model systems [21]–[24]. Therefore, we hypothesized that inhibiting NF-κB activity in the A549.I cell line would dampen EMT induction. Immunoblot analysis confirmed that A549.I cells fail to down regulate E-cadherin expression or upregulate mesenchymal markers (Vimentin, N-cadherin and Fibronectin) compared to control cells (Figure 6A). Moreover, cytokine-treated A549.I cells showed only minimal upregulation of TWIST1, ZEB2 and SNAI2 gene expression following TNF and TGFβ treatment (Figure 6B). These results indicate that NF-κB is required to upregulate TWIST1, ZEB2 and SNAI2, while expression of SNAI1 appears independent of NF-κB-dependent transcription in the A549.I cell line. These results suggest that the expression of critical EMT master-switch transcription factors requires NF-κB activity.

Figure 6. NF-κB is required for the maintenance of CICs and lung metastasis.

(A) A549.I cells fail to show changes in mesenchymal markers, as determined by immunoblot analysis. (B) NF-κB is required to upregulate mRNA expression of master-switch transcription factors. (C) Spheroid cultures of A549 and H157 cell lines, expressing empty vector or the Flag-IκB super-repressor, were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. The cells were disaggregated and subjected to invasion assays. (D) A549.V and A549.I 3D cultures were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. The cells were disaggregated and SC injected into nude mice (1×106animal). Forty days later, animals were sacrificed and the number of surface lung metastasis were determined. In addition, SC tumors were excised and wet tumor weight determined. Weight and lung metastases data from Figure 6 are mean ± S.D. of five mice per condition, *p<0.05, N = 3 independent experiments. The graphs in Figure 6 are mean ± S.D., *p<0.05, of three independent experiments. Data with P values greater than 0.05 were considered not significant (ns). QRT-PCR experiments are normalized to GAPDH expression.

Next, we assessed whether NSCLC required NF-κB for invasion using transwell assays. Inhibited NF-κB activity in A549.I cells abolished invasion through Matrigel when compared to the control lines (Figure 6C). This effect was not cell-line specific since another NSCLC line expressing the SR-IκB (H157.I) showed similar results as A549.I cells. Because data shown in Figure 6 indicate that NF-κB is required for NSCLC to undergo EMT, we tested the A549.V and A549.I cell for their ability to metastasize to lung using a nude mouse model. As expected, cytokine-treated A549.I cells failed to form lung metastases (Figure 6D, left). The inability of these cells to metastasize to lung was not due to a loss of cell viability or an inability to form primary tumors, since untreated A549.I formed SC tumors with similar growth rates as A549.V cells (Figure 6D, right). Thus, data shown in Figure 6 indicates that TNF and TGFβ treated 3D NSCLC cultures require NF-κB to upregulate master-switch transcription factors, induce EMT, and promote invasive properties. Moreover, without NF-κB transcriptional activity A549 cells lose their ability to metastasize to lung without impacting primary tumor growth.
Discusson NF-κB regulates EMT to potentiate metastatic progression of NSCLC
We implemented a simple and relatively quick 3D culture system to examine the importance of NF-κB signaling during EMT induction and CIC propagation within NSCLC cell lines. In response to TNF and TGFβ exposure, A549 spheroid cultures displayed a loss of E-Cadherin and elevated expression of mesenchymal markers, N-Cadherin, Vimentin, and Fibronectin. The increased expression of mesenchymal protein markers likely occurs due to induction of the EMT master-switch transcription factors, TWIST1, SNAI1, SNAI2 and ZEB2. Furthermore, spheroid populations of mesenchymal A549 cells show elevated expression of endogenous transcription factors known to potentiate dedifferentiation, including KLF4, SOX2, POU5F1, MYCN and KIT. Interestingly, mesenchymal A549 cells from spheroid cultures failed to generate large SC tumors, compared to 2D cultures. Despite this effect, cytokine-treated 3D A549 cells displayed elevated lung surface metastatic lesions. These results support the hypothesis that CICs extravasated into the circulatory system and metastasize to the lung without forming SC tumors. We further demonstrated that EMT-induced A549 3D cultures effectively metastasize to lung under limiting cell dilutions, confirming the presence of an enriched “stem-like” CIC population. Thus, our results suggest that EMT induction effectively selects for self-renewing CIC with metastatic potential, a phenotype described by Dieter and colleagues as self-renewing long-term tumor initiating cells responsible for color cancer metastasis [51]. Since IKK and NF-κB pathways have been linked to EMT and development of CICs [21]–[25], [31], we examined whether mesenchymal A549 cells upregulate NF-κB transcriptional activity. Surprisingly, EMT-induced spheroid A549 cultures displayed chronic IKK activity as measured by phosphorylation of IκBα(pS32) and RelA(pS536), and by constitutive expression of IL8 and BIRC3 transcripts. Moreover, cytokine-treated spheroid A549 cultures maintained the activation of IKK signaling pathways well beyond the half-life of the TNF and TGFβ cytokines added to the culture media. These results suggest that mesenchymal A549 spheroid cultures must produce autocrine factors capable of maintaining the EMT phenotype. Importantly, constitutive NF-κB activity proves essential for effective EMT partially through its ability to upregulate the master-switch transcription factors TWIST1, ZEB2 and SNAI2. As a result, the loss of NF-κB activity prohibited cytokine-treated spheroid A549 cells from becoming invasive and also abolished lung metastasis in the mouse xenograft model. This work firmly establishes a role for NF-κB in the induction of EMT and for the development of NSCLC CICs that promote metastasis.
Spheroid models and the propagation of CICs
Various 3D culture models have been developed that more accurately mimic tumor biology, such as cell-cell contacts, extracellular matrix composition, and nutrient accessgradients [52]–[54]. The advantage to using the hanging drop technique, over other techniques, is that 2D cultures can be quickly expanded to form multicellular aggregates that share similar size and shape, and mesenchymal populations are generated within six days. Data provided in Figure 1C demonstrate that multiple NSCLC cell lines form compact spheroids that undergo highly reproducible EMT when exposed to TNF and TGFβ. Moreover, these spheroids possess increased sensitivity to TNF and TGFβ compared to monolayer cultures (Figures 2, 3, and 4). Therefore, we utilized this 3D system to examine EMT and CIC formation in NSCLC cell lines. Surprisingly, A549 spheroids show increased migration without requiring exposure to TNF and TGFβ and despite expressing epithelial markers (Figure 1B, 2A, and 3A). This indicates that phenotypic changes occur in 3D cultures prior to exposure to EMT-inducing cytokines. However, increased invasion is restricted to cytokine-induced A549 spheroid cultures and corresponds with the upregulation of matrix and extracellular remodeling enzymes known to induce invasive properties [41], [42]. Therefore, spheroid cultures are poised to respond to TNF and TGFβ cytokines and are able to sustain EMT reprogramming. Together, we establish that CIC populations formed from EMT induction of 3D NSCLC cell lines provide a useful tool for further characterization of cancer progression in the lung.
Mesenchymal A549 cells show constitutively active NF-κB signaling pathways
Constitutive NF-κB activation occurs in many different types of hematopoietic and epithelial-derived carcinomas. However, mutations that result in chronic activation of NF-κB signaling are extremely rare in epithelial cancers [19]. Thus, activation of NF-κB most likely results from autocrine and paracrine signaling within the tumor microenvironment rather than genetic alterations [2], [19]. Our data support this hypothesis by showing that TNF- and TGFβ-treated A549 spheroid populations both undergo EMT and maintain constitutive NF-κB signaling (Figure 5A and 5B). Rather than using co-culture systems, which introduce contaminating cell types other than NSCLC cells, we chose to treat A549 spheroids with EMT-inducing cytokines. TNF and TGFβ were selected because within the tumor microenvironment, TNF is believed to be produced predominantly by tumor-associated macrophages, while TGFβ is secreted by fibroblast and endothelial cells. This combination of cytokines not only effectively and reproducibly induces EMT, but also facilitates a reprogramming event that results in chronic NF-κB signaling. The molecular mechanism by which this occurs is currently unknown, but most likely is due to an increase in NF-κB-regulated gene products that function in an autocrine-dependent manner to maintain active NF-κB.
Inflammatory regulatory circuits that drive constitutive NF-κB activation
In the past two years, evidence has emerged that an epigenetic switch occurs during breast cancer transformation in which inflammatory circuits involving IL6 and IL8 mediate self-renewal of CICs [55]–[57]. Ginesteir and colleagues showed that breast CICs upregulate the IL8 receptor CXCR1 to potentiate self-renewal, tumorigenicity and metastasis [57]. Additional studies indicate that oncogenic transformation of breast cancer cells leads to chronic activation of NF-κB required to upregulate Lin-28B and downregulate the negative microRNA regulator of IL6, Let-7a [56]. As a result, IL6 provides an inflammatory feedback loop that further activates NF-κB as well as the STAT3 signaling pathway [56], [58]. Interestingly, this pro-inflammatory feedback loop also exists in some prostate and hepatocellular carcinomas, but only a subset of lung cancers showed increased IL6 expression [56]. In addition to the autocrine feedback mechanism, IL6 signaling pathways downregulate mir200c in a chemically-induced transformed breast cancer cell line. Loss of mir200c subsequently results in constitutively activated NF-κB through an inflammatory feedforward signaling circuit [59]. In these papers [55], [56], [59], IL6 was found to be required for the maintenance of breast CICs.

Additional work is needed to determine the importance of IL8 and IL6 as feedforward mediators of NF-κB activation in mesenchymal NSCLC cell lines. As shown in Figure 5B, IL8 is highly upregulated and maintained in mesenchymal A549 cultures; however, IL6 transcripts do not significantly change between untreated 3D and cytokine-treated 3D cultures (Figure S2C). Thus, in agreement with IIiopoulos and colleagues [59], IL6 may not be a common requirement for CICs in lung cancer. However, since CXCR1 is highly expressed in A549 cells following exposure to DNA methytransferase inhibitors [60], inflammatory circuits that regulate promoter demethylation, as observed for IL6 signaling [59], may play an important role for controlling the IL8CXCR1 responsiveness in lung cancers. Future work is needed to explore the importance of IL8CXCR1 in the maintenance of constitutive NF-κB activation and development of NSCLC CICs.
Supporting Information Figure S1.
Cytokine-treated 3D A549 cells show increased fibroid and mesenchymal morphology. Monolayer (2D) and 3D A549 cultures were left alone or treated with TNF and TGFβ for ninety-six hours. Cells were subsequently disaggregated, replated on glass coverslips, and cultured for an additional eighteen hours in 2% FBS. The cells were then fixed in methanol, and indirect immunofluorescence was used to detect the presence of junctional E-cadherin. Images are a representative field from three independent experiments.

Figure S2.
TNF and TGFβ-treated 3D A549 cells show increased RelA phosphorylation and nuclear DNA binding activity. (A) Immunoblot analysis of 3D A549 cells indicates that cells display constitutive RelA phosphorylation upon co-stimulation with both TNF and TGFβ over the three day period. (B) Nuclear extracts from cytokine-treated 3D control A549.V cells show elevated NF-κB binding activity by EMSA, compared to unstimulated cell extracts. The NF-κB DNA-protein complex is composed of both RelA and p50 proteins as detected by antibody super shift (SS) assays. (C) In contrast to IL8 expression shown in Figure 5B, cytokine-treated 3D cultures fail to upregulate IL6 transcripts as measured by QRT-PCR.

Table S1.
QRT-PCR Primers.

Conceived and designed the experiments: MWM MK DFA NNB JJW. Performed the experiments: MK DFA NNB JJW. Analyzed the data: MWM MK DFA NNB JJW. Contributed reagentsmaterialsanalysis tools: AJK SB DRJ. Wrote the paper: MWM MK DFA JJW.
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FTP only AFP only SMB only AFT and FTP AFP and SMB Answer: E Question: 80 Network users often have multiple passwords, a distinct password for each network service they access. Which is NOT a valid way to simplify this situation for users in Mac OS X v10.3? Set up a Kerberos environment on the network. Have users store their login information for different servers in Keychain. Set up a directory service to make user and password information available to all computers. Have users access network servers without authenticating, using the Network icon in the Finder-rather than using the Connect Server command, which requires users to authenticate. Answer: D Question: 81 Which is an appropriate FIRST step when performing a software update on multiple computers That have Mac OS X v10.3? Restart each computer in single-user mode. On one computer, choose Download Checked items from the update menu in Software update. On one computer, choose copy update to multiple computers from the update menu in software update. Copy the Software Update preferences from a computer on which the update has been installed to all the other computers. Answer: B Question: 82 You are in a Terminal window, and your current directory is your home directory. What could you Type to display the contents of the file named my file? Which resides in your home directory? cat My File cat /Myfile cat~MyFile cat./MyFile cat ~/MyFile Answer: A, D, E Question: 83 Which statements are true about Open Directory? Choose all that apply Open Directory is extensible via plug-ins. Open Directory runs on Mac OS X Server only. Open Directory does NOT provide load balancing. You manage Open Directory using Open Directory Assisyant. An Open Directory server can function as a windows Primary Domain Controller. Answer: A, E Question: 84 What does the Console utility do? Displays log files Controls monitor setup. 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