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Download references. We thank C. Roy Yale University for providing the RavZ expression plasmids, B. Levine UT Southwestern for providing tissues from beclin 1-knockout mice, and A. Darehshouri at UT Southwestern Electron Microscopy Core Facility for training in electron microscopy sample preparation and image processing.

We are grateful for suggestions and technical supports from Chen Laboratory members, especially L. Sun, Y. Wu and S. This work was supported by grants from the Cancer Prevention and Research Institute of Texas RP and RP and the Welch Foundation I was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health T32AI The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

and X. are Cancer Research Institute Irvington Postdoctoral Fellows. is an Investigator of Howard Hughes Medical Institute. Nature thanks Kate Fitzgerald and the other anonymous reviewer s for their contribution to the peer review of this work.

Department of Molecular Biology, Center for Inflammation Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA. You can also search for this author in PubMed Google Scholar. and H. planned the study under the guidance of Z.

performed the mass spectrometry experiments. and F. generated several cell lines used in this study. designed and supervised this study. and Z. prepared and revised the manuscript.

Correspondence to Zhijian J. a , DNA and cGAMP, but not RNA, trigger LC3 lipidation. BJ cells were stimulated with cGAMP by digitonin permeabilization or transfected with ISD or poly I:C.

Cell lysates were analysed by immunoblotting with the indicated antibodies. b , DNA virus but not RNA virus induces LC3 conversion. BJ cells were infected with wild-type WT HSV-1, HSV-1 ΔICP c , cGAMP induces STING degradation in the lysosome.

HeLa cells that stably express STING—Flag were treated with cGAMP or starved in the presence or absence of chloroquine, followed by immunoblotting. d , Inhibition of TBK1 or IKK does not impair LC3 lipidation.

Inhibitors of TBK1 BX or MRT or IKK TPCA1 were incubated with BJ cells before stimulation of the cells with cGAMP. Cell lysates were analysed by immunoblotting. e , Control experiment showing that TPCA1 inhibits IκBα degradation by inhibiting IKK.

The cells were stimulated with cGAMP as indicated. Mean ± s. is shown. g , STING S phosphorylation by TBK1 is essential for IRF3 phosphorylation but not LC3 conversion. h , HeLa cells stably expressing LC3—GFP and different STING mutants RA, YA or VM were stimulated with cGAMP followed by confocal immunofluorescence microscopy.

i , Quantification of the cells with colocalization of LC3 and STING puncta. j , k , HeLa cells that stably express LC3GFP and STING—Flag were treated with bafilomycin A1 BafA1 or BFA followed by stimulation with cGAMP and confocal immunofluorescence microscopy.

l , BJ cells were treated with BFA, lysosome inhibitors BafA1 or chloroquine or proteasome inhibitors MG or Velcade before stimulation with cGAMP.

a , Expression plasmids that encode truncated STING mutants were transiently transfected into HEKT cells for 24 h, followed by stimulation with cGAMP for 4 h. b , Expression plasmids of indicated STING truncation mutants were transfected into HeLa LC3—GFP cells for 24 h.

The cells were stimulated with cGAMP followed by immunostaining and fluorescence microscopy. c , d , Mutations of STING at L and R impaired LC3 lipidation and TBK1 activation.

c , Expression plasmids that encode full-length STING containing the indicated mutations were transiently transfected into HEKT cells, followed by stimulation with cGAMP. FL, full length. d , Indicated STING mutants were transfected and stimulated as described in c followed by immunostaining and fluorescence microscopy.

e , Schematic of functional domains and residues of human huSTING and sea anemone STING nvSTING , highlighting the evolutionary conservation of the cyclic dinucleotide CDN binding domain but not the C-terminal activation domain.

b , Expression vectors encoding human cGAS hu-cGAS , Nv cGAS or DncV were transfected into HEKT cells for 24 h. Small molecules were extracted from cells for analysis of cGAMP isomers by tandem mass spectrometry. Mass spectra of cGAMP from Nv cGAS-expressing cells match those from hu-cGAS-expressing cells but not those from DncV-expressing cells.

c , Similar to b , except that the small-molecule extracts were fractionated using a monoQ column, and each fraction was delivered into THP1—ISG luciferase reporter cells for measuring the CDN activity. d , CDNs produced by hu-cGAS and Nv cGAS, but not that by DncV, is resistant to digestion by RNase T1.

Small-molecule extracts from HEKT cells expressing the indicated CDN synthases were treated with RNase T1 or RNaseT2 or left untreated before delivery into THP1—ISG luciferase reporter cells to measure the CDN activity.

e , Domain organization of STING from human hu , Danio rerio Dr and Xenopus tropicalis Xt. f , Xenopus STING stimulates LC3 lipidation but not IRF3 phosphorylation. HEKT cells were transfected with expression plasmids for STING—Flag from human, Danio or Xenopus for 24 h and then stimulated with cGAMP for 1 h.

Anti-Flag antibody was used to immunoprecipitate STING from cell lysates followed by immunoblotting with the indicated antibodies. g , cGAMP stimulates formation of perinuclear puncta of STING from different species. HeLa cells transiently expressing human, Danio or Xenopus STING—Flag were stimulated with cGAMP for 3 h.

Cells were immunostained with a Flag antibody followed by fluorescence microscopy. h , Quantification of the percentage of cells with STING peri-nuclear foci formation. All results in this figure are representative of at least two independent experiments.

a , STING colocalizes with the ERGIC and autophagosomes in response to cGAMP stimulation. HeLa cells that stably express GFP—LC3 and STING—Flag were stimulated with cGAMP for the indicated time followed by immunofluorescence microscopy.

b , STING trafficking to the ERGIC is blocked by GCA. BJ cells were stimulated with cGAMP for 3 h in the presence or absence of GCA. Cells were stained with DAPI or the indicated antibodies and examined by confocal microscopy.

c , Procedures for in vitro reconstitution of cGAMP-induced LC3 lipidation and membrane fractionation. Membrane pelleted at 25, g P25 from these cells was incubated with cytosolic extracts S from starved or untreated T cells in the presence of GTP and an ATP regenerating system.

After incubation at 30 °C for 90 min, the reaction mixtures were analysed by immunoblotting. e , Similar to c , except that different organelle membranes enriched by differential centrifugation were prepared and incubated with cytosol S from HEKT cells to detect LC3 lipidation. f , Similar to c , except that P25 membranes were further fractionated by sucrose ultracentrifugation to generate P25P pellet and P25L light and incubated with cytosol S from HEKT cells to detect LC3 lipidation.

a , BJ cells were stimulated with cGAMP for the indicated time. Cells were immunostained with a STING antibody together with an antibody against GM cis -Golgi , TGN38 trans -Golgi , GGA3 post-Golgi vesicles , CD63 late endosomes or LAMP1 lysosomes , followed by immunofluorescence microscopy.

b , Quantification of the percentage of cells in which STING colocalized with different organelle markers in a.

c , cGAMP induces trafficking of STING to the ERGIC, COP-I vesicles and LC3 autophagosomes. HeLa cells that stably express STING—Flag and LC3—GFP were stimulated with cGAMP for the indicated time.

Cells were immunostained with antibodies specific for Flag to detect STING , ERGIC53 ERGIC or beta-COP COP1 vesicles , followed by fluorescence microscopy. d , Quantification of the percentage of cells in which STING colocalized with different organelle markers in c.

e , BJ cells were stimulated with cGAMP in the presence or absence of bafilomycin A1 BafA1. Cells were immunostained with an antibody specific for STING or RAB7A followed by microscopy. f , Quantification of the percentage of cells in which STING colocalized with RAB7A in e. g , BJ cells were transfected with an siRNA targeting RAB7A or a control siRNA for three days before stimulation with cGAMP for the indicated time.

a , ATG5 is required for LC3 lipidation but dispensable for STING degradation induced by cGAMP. b , ATG9 is dispensable for LC3 lipidation and STING degradation. c , STING activation does not induce mTOR inhibition. BJ cells were treated with cGAMP, HT-DNA, torin 1 or rapamycin for the indicated time followed by immunoblotting of cell lysates.

d , Loss of ULK1 and ULK2 impairs LC3 conversion and p62 degradation induced by mTOR inhibition. e , STING-induced LC3 conversion is independent of ULK1 and ULK2. Boxed areas are enlarged to show double-membrane organelles that represent autophagosomes.

Red arrows highlight double-membrane characteristic of autophagosomes in stimulated cells. Scale bar, 1 µm original image , nm magnified image. a , BECN1 is dispensable for LC3 conversion triggered by cGAMP.

b , BECN1 is not essential for LC3 conversion in conventional autophagy. c , VPS34 depletion delayed cGAMP-induced STING degradation but not LC3 lipidation. VPSknockout BJ cells were treated with cGAMP for the indicated time followed by immunoblotting of cell lysates.

Red arrow highlights double-membrane characteristic of autophagosomes in stimulated cells. e , f , ULK1 and VPS34 are essential for LC3 puncta formation induced by torin 1 but not by cGAMP.

LC3—GFP puncta formation was visualized by fluorescence microscopy e and the percentage of cells with LC3—GFP peri-nuclear foci formation was quantified f. Yui and H. Harashima, Chem. To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

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Issue 50, From the journal: Chemical Communications. jp b Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai , Chiyoda-ku, Tokyo , Japan E-mail: tamura.

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Oncologist 19 6 , — Wu, T. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer.

In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Autophagy is a degradative program that maintains cellular homeostasis. Autophagy defects have been described in numerous diseases. However, analysis of autophagy rates can be challenging, particularly in rare cell populations or in vivo, due to limitations in currently available tools for measuring autophagy induction.

Here, we describe a method to monitor autophagy by measuring phosphorylation of the protein ATG16L1. We developed and characterized a monoclonal antibody that can detect phospho-ATG16L1 endogenously in mammalian cells.

Importantly, phospho-ATG16L1 is only present on newly forming autophagosomes. Therefore, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can confound autophagy analysis.

Moreover, we show that ATG16L1 phosphorylation is a conserved signaling pathway activated by numerous autophagy-inducing stressors. The described antibody is suitable for western blot, immunofluorescence and immunohistochemistry, and measured phospho-ATG16L1 levels directly correspond to autophagy rates.

Taken together, this phospho-antibody represents an exciting tool to study autophagy induction. This is a preview of subscription content, access via your institution.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Parzych, K. An overview of autophagy: morphology, mechanism, and regulation. Redox Signal.

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