Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why Submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research

User menu

  • My alerts

Search

  • Advanced search
Life Science Alliance
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research
  • My alerts
Life Science Alliance

Advanced Search

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why Submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Follow lsa Template on Twitter
Research Article
Source Data
Transparent Process
Open Access

Attenuation of cGAS/STING activity during mitosis

Brittany L Uhlorn, Eduardo R Gamez, Shuaizhi Li, View ORCID ProfileSamuel K Campos  Correspondence email
Brittany L Uhlorn
1Cancer Biology Graduate Interdisciplinary Program, The University of Arizona, Tucson, AZ, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eduardo R Gamez
2Department of Physiology, The University of Arizona, Tucson, AZ, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shuaizhi Li
3Department of Immunobiology, The University of Arizona, Tucson, AZ, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samuel K Campos
1Cancer Biology Graduate Interdisciplinary Program, The University of Arizona, Tucson, AZ, USA
3Department of Immunobiology, The University of Arizona, Tucson, AZ, USA
4BIO5 Institute, The University of Arizona, Tucson, AZ, USA
5Department of Molecular and Cellular Biology, The University of Arizona, Tucson, AZ, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Samuel K Campos
  • For correspondence: skcampos@email.arizona.edu
Published 13 July 2020. DOI: 10.26508/lsa.201900636
  • Article
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF
Loading

Article Figures & Data

Figures

  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1. Human keratinocytes respond to foreign DNA via cGAS/STING.

    HaCaTs were transfected with siRNAs for 16 h, followed by transfection with 500 ng pGL3 or water for 90 min. Lysates were analyzed for cGAS/STING component knockdown and pathway activity by SDS–PAGE and Western blot.

    Source data are available for this figure.

    Source Data for Figure 1[LSA-2019-00636_SdataF1.tif]

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2. Effects of Golgi disruption on DNA-dependent subcellular localization of STING and IRF3.

    HaCaT cells were pretreated with vehicle, NOC, GCA, or BFA before a 90-min transfection with H2O or pGL3. (A, B) Cells were fixed and stained for (A) STING and p230 or (B) IRF3 and TGN46 before DAPI staining. (A, B) Representative micrographs are shown in (A, B), white arrowheads indicate overlap. (C, D) Manders’ overlap coefficients from multiple micrographs were plotted for (C) STING:p230 and (D) IRF3:DAPI. *P < 0.01, ***P < 0.0001. Scale bars = 10 μm.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3. Effects of Golgi disruption on cGAS/STING activity.

    (A) Cells were treated with vehicle, nocodazole (NOC), golgicide A (GCA), or brefeldin A (BFA) for 1 h prior and during a 90-min transfection with HSV-60 oligonucleotide, calf-thymus DNA (CTD), or plasmid pGL3. (B, C, D) Transfection of vehicle- or GCA-treated cells with pGL3 or pIC, and (C, D) densitometric quantification of pIRF3 blots. **P < 0.001, n = 5 biological replicates. (E) Vehicle- or GCA-treated cells were treated with H2O or 12.5 μg cGAMP for 90 min before SDS–PAGE and Western blot for cGAS/STING pathway components. (F) cGAMP production in vehicle- and GCA-treated cells upon pGL3 transfection. n = 3 biological replicates, with n = 2 technical replicates each.

    Source data are available for this figure.

    Source Data for Figure 3[LSA-2019-00636_SdataF3.tif]

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4. Golgi vesiculation impairs DNA-dependent IFN, ISG, and chemokine gene transcription.

    HaCaT cells were treated with GCA or vehicle for 1 h before a 90-min pGL3 transfection. (A, B, C, D, E, F, G, H) Transcript levels of (A) IFNB1, (B, C, D, E, F) ISGs, and (G, H) chemokine genes were measured via RT-qPCR and normalized to TATA-binding protein. *P < 0.01, **P < 0.001, ***P < 0.0001, for comparisons of 4 and 8 h DMSO-treated pGL3 groups to 4 and 8 h DMSO-treated H2O controls and for 4 and 8 h DMSO-treated pGL3 groups to 4 and 8 h GCA-treated pGL3 groups, n = 3 technical replicates.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5. cGAS/STING activity is attenuated during mitosis.

    (A) Cell synchronization method. Cells were pre-synchronized in G1 by contact inhibition and growth in low serum. Cells were then released by replating at subconfluence in 10% serum and synchronized in prometaphase with NOC. Cell cycle analysis by propidium iodide staining. Cells were transfected with pGL3 for 90 min at various times post-release from NOC. (B) pIRF3 activation in response to pGL3 was dampened when arrested cells were transfected, gradually returning as cells completed mitosis and returned to interphase. (C) Densitometric quantification of relative pIRF3 increase in response to pGL3 transfection across multiple blots. *P < 0.05, for comparison of 180 min to NR groups, n = 4 independent biological replicates. (D) Golgi localization of STING was impaired when arrested cells (NR) were transfected, but was restored upon return to interphase (180 min). (E) Manders’ overlap coefficients from multiple micrographs were plotted for STING:p230, **P < 0.001, scale bars = 10 μm.

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6. cGAS and STING are nonresponsive during mitosis.

    (A) Mitotic phospho-IRF3 and phospho-STING responses to pGL3. Asynchronous and arrested cells were stimulated by transfection of 500 ng pGL3 or water for 90 min before Western blots. (B) Mitotic cGAMP responses to pGL3. Asynchronous and arrested cells were transfected with 500 ng pGL3. Non-internalized transfection complexes were removed by media change 2 h after the initial transfection. 5 h post-transfection, cGAMP levels were measured by ELISA. *P < 0.05, n = 2 biological replicates, with n = 2 technical replicates each. (C, D) cGAS subcellular localization and Golgi morphology in asynchronous (C) and arrested (D) HaCaT cells. Scale bars = 10 μm. (E) Mitotic phospho-IRF3 and phospho-STING responses to exogenous cGAMP. Asynchronous and arrested cells were treated with 25 μg/ml 2′-3′cGAMP for 2 h before Western blots.

    Source data are available for this figure.

    Source Data for Figure 6[LSA-2019-00636_SdataF6.tif]

PreviousNext
Back to top
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word on Life Science Alliance.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Attenuation of cGAS/STING activity during mitosis
(Your Name) has sent you a message from Life Science Alliance
(Your Name) thought you would like to see the Life Science Alliance web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Dampening of both cGAS and STING activity during mitosis
Brittany L Uhlorn, Eduardo R Gamez, Shuaizhi Li, Samuel K Campos
Life Science Alliance Jul 2020, 3 (9) e201900636; DOI: 10.26508/lsa.201900636

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Dampening of both cGAS and STING activity during mitosis
Brittany L Uhlorn, Eduardo R Gamez, Shuaizhi Li, Samuel K Campos
Life Science Alliance Jul 2020, 3 (9) e201900636; DOI: 10.26508/lsa.201900636
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
Issue Cover

In this Issue

Volume 3, No. 9
September 2020
  • Table of Contents
  • Cover (PDF)
  • About the Cover
  • Masthead (PDF)
Advertisement

Jump to section

  • Article
    • Abstract
    • Introduction
    • Results and Discussion
    • Materials and Methods
    • Acknowledgements
    • References
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF

Subjects

  • Cell Biology
  • Immunology

Related Articles

  • No related articles found.

Cited By...

  • No citing articles found.
  • Google Scholar

More in this TOC Section

  • NLRP3 controls ATM activation
  • RIF1 stabilizes replication domains
  • Chromatin context-dependent interferon response
Show more Research Article

Similar Articles

EMBO Press LogoRockefeller University Press LogoCold Spring Harbor Logo

Content

  • Home
  • Newest Articles
  • Current Issue
  • Archive
  • Subject Collections

For Authors

  • Submit a Manuscript
  • Author Guidelines
  • License, copyright, Fee

Other Services

  • Alerts
  • Twitter
  • RSS Feeds

More Information

  • Editors & Staff
  • Reviewer Guidelines
  • Feedback
  • Licensing and Reuse
  • Privacy Policy

ISSN: 2575-1077
© 2023 Life Science Alliance LLC

Life Science Alliance is registered as a trademark in the U.S. Patent and Trade Mark Office and in the European Union Intellectual Property Office.