Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 1. STING activation elevates pH in Golgi-derived vesicles, leading to LC3 lipidation through V-ATPase and ATG16L1. (A) HeLa-STING(WT), HeLa-STING- ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h, or 100 nM Torin1 for 5 h, or untreated. The cell lysates were analyzed by immunoblotting as indicated. (B) HEK293T FIP200-KO cells stably expressing STING and GFP-LC3B were stimulated with indicated compounds (30 µM MSA2, 100 nM bafilomycin A1) for 2 h, or untreated. Cells were analyzed by confocal microscopy. (Scale bar, 10 µm.) (C) HeLa-STING-ATG16L1 and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 1.5 h or untreated. Protein complexes were immunoprecipitated with a Flag antibody and analyzed by immunoblotting. (D) HeLa cells stably expressing STING, miRFP670-LC3B, and Super Ecliptic pHluorin (SEP)-mKate2-TGN46 were treated with 1 µM diABZI, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min, 18 min, and 27 min are shown. The Left panels show the SEP signal of SEP-mKate2-TGN46 fusion protein. (Scale bar, 10 µm) for images on the Upper panels and 1 µm for Zoom-in images on the Bottom panels.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Derivative Assay, Western Blot, Stable Transfection, Expressing, Confocal Microscopy, Immunoprecipitation, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 2. Noncanonical autophagy is required for STING-induced activation of MiT/TFE transcription factors. (A) HeLa-STING (ctrl) and HeLa-STING-KO cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h or untreated, followed by analyses of cell lysates by immunoblotting with the indicated antibodies. (B) HeLa- STING cells stably expressing TFEB-miRFP670 were treated with 50 nM diABZI for 2 h or untreated, and live cells were imaged by microscopy. (Scale bar, 10 µm.) (C) Quantification of 77 and 80 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING, HeLa-STING-ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h or 100 nM Torin1 for 5 h or untreated. The cell lysates were analyzed by immunoblotting as indicated. (E) HeLa cells stably expressing STING (ctrl) or both STING and SopF (SopF) were treated with 30 µM MSA2 for 2 h or untreated. The cell lysates were analyzed by immunoblotting. (F) HeLa-STING, HeLa-STING-SopF, and HeLa-STING-ATG16L1KO cells stably expressing SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. The cells were stained with an antibody against endogenous TFEB followed by confocal microscopy analyses. (Scale bar, 10 µm.) (G) Quantification of 88 to 228 cells per group as represented by F. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Microscopy, Staining, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 3. TFEB activation by STING does not require TBK1. (A) HeLa-STING and HeLa-STING (1 to 370) cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (B) HeLa-STING (1 to 370) cells were stimulated with 30 µM MSA2 for 2 h or untreated; then, cells were imaged by confocal microscopy using an antibody against TFEB. NucBlue stains nuclear DNA. (Scale bar, 10 µm.) (C) Quantification of 67 and 77 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING (1 to 370), and HeLa-STING (1 to 370)-SopF cells were treated with 10 µM MSA2 for 24 h or untreated. RNA was isolated from these cells for analysis by quantitative RT-PCR to measure the expression of indicated TFEB target genes. CTSD: cathepsin D; CTSB: cathepsin B; GPNMB: Glycoprotein Nonmetastatic Melanoma Protein B. The mRNA expression levels of the MSA-treated cells were normalized to untreated cells (ctrl). Error bars represent SD. The data are representative of 2 independent experiments. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) wild-type (WT) and TBK1/IKKε double knockout (DKO) THP1 cells were treated with 30 µM MSA2 for 2 h or untreated. Then cell lysates were analyzed by immunoblotting.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Confocal Microscopy, Isolation, Quantitative RT-PCR, Expressing, Double Knockout

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 4. STING agonist C53 activates TBK1 and IRF3, but not TFEB, through a mechanism independent of membrane trafficking. (A) HeLa-STING cells were treated with 30 µM MSA2, 50 nM diABZI, or 10 µM C53 for 2 h or untreated, then cell lysates were analyzed by immunoblotting. (B) HeLa STING-KO cells stably expressing STING-mGFP and mCherry-Sec61B were treated with 1.25 µM C53, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min (Ctrl) and 10 min (C53) are shown. (Scale bar, 10 µm) for images on the Left panels and 2 µm for Zoom-in images on the Right panels. (C) HeLa-STING cells were treated with 1 mM BFA or 10 mM GCA for 20 min before stimulation with 50 nM diABZI or 10 µM C53 for 2 h. Cell lysates were analyzed by immunoblotting with indicated antibodies. (D) HeLa-STING cells were stimulated with 1.25 µM C53 for 20 min or untreated, then cells were imaged by confocal microscopy using antibodies against STING and p-TBK1. NucBlue stains nuclear DNA. (Scale bar, 10 µm.)

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Membrane, Western Blot, Stable Transfection, Expressing, Microscopy, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 5. FNIP1/FNIP2–GABARAP interactions mediate the activation of TFEB by STING. (A) HeLa-STING (Ctrl), HeLa-STING-FNIP1/2DKO, HeLa-STING-FNIP1WT, and HeLa-STING-FNIP1MUT cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting as indicated. (B) HeLa-STING (WT), HeLa-STING-LC3sTKO, and HeLa-STING-GABARAPsTKO cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (C) Similar to (B) except that cells were analyzed by immunofluorescent microscopy using an antibody against TFEB. TFEB+NucBlue: merging of TFEB staining with nuclear DNA staining by NucBlue. (Scale bar, 10 µm.) (D) Quantification of 46 to 76 cells per group as represented by C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) HeLa-STING cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h, or 100 nM Torin1 for 5 h, or untreated. Cell lysates were analyzed by immunoblotting. (F) Diagram depicting the mechanism by which STING-induced noncanonical autophagy promotes endolysosomal biogenesis through inhibiting the phosphorylation of TFEB, TFE3, and MITF by mTORC1.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Microscopy, Staining, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 7. STING-induced autophagy recruits the ALIX–ESCRT to mitigate endolysosomal perturbation. (A) HeLa-STING, HeLa-STING-SopF, and HeLa-STING- ATG16L1KO cells that stably express SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. Cells were imaged by microscopy using an antibody against ALIX and other markers as indicated. (Scale bar, 10 µm.) Arrows indicate ALIX puncta. (B) Quantification of 32 to 70 cells per group represented by A. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (C) HeLa-STING cells transfected with siRNA against ALIX or control siRNA were treated with 50 nM diABZI for 24 h, and the morphology of the cells was analyzed by bright-field microscopy. (Scale bar, 25 µm.) (D) Quantification of 228 and 214 cells per group shown in C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ***P < 0.001. (E) HeLa-STING-ATG16L1 and HeLa-STING- ATG16ΔWD40 cells treated with 50 nM diABZI for 24 h or untreated, then cells were analyzed by bright-field microscopy. (Scale bar, 25 µm.) (F) Quantification of more than 300 cells per group shown in E. Error bars represent SD. Tested by unpaired Student’s t test. ***P < 0.001. (G) A Diagram showing that GABARAPs lipidation facilitates ALIX-mediated ESCRT recruitment which mitigates endolysosomal perturbation.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Stable Transfection, Microscopy, Transfection, Control