STING inhibits autophagosome–lysosome fusion. (A) Autophagic flux is accelerated in STING-KO MEFs compared with WT MEFs. WT MEFs and STING-KO MEFs were treated with Torin 1 with or without CQ for the indicated time and then immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (B) Inducible expression of STING at endogenous level inhibits autophagic flux in MEFs. Inducible stable cell line expressing STING was treated with different doses of doxycycline (0, 100, 200 ng/ml) for 24 h to induce STING expression. After 24 h, WT MEFs, STING-KO MEFs, and STING-KO MEFs reconstituted with inducible STING were treated with or without Torin 1 for 3 h. Cells were lysed and immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (C) Fusion between autophagosomes and lysosomes is promoted in STING-KO MEFs. WT MEFs, STING-KO MEFs, and STING-KO MEFs reconstituted with STING-HA were treated with Torin 1 for 3 h before fluorescence of GFP-RFP-LC3 was imaged. Scale bar, 5 µm. (D) Quantification of the results in C. Percentage of RFP + GFP-dots in total RFP dots. Data are mean ± SD for 100 cells. ***, P ≤ 0.001. (E) Expression of STING-HA inhibits the autophagic flux in HEK293T cells. HEK293T cells were transfected with empty vector, WT STING-HA. 24 h after transfection, cells were treated with Torin 1 combined with or without CQ for indicated time. Immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (F and G) STING regulated autophagosome–lysosome fusion is STX17 (F), ULK1, ATG9, ATG14, and ATG5 (G) dependent. Immunoblot analysis of LC3-II conversion and p62 degradation in MEFs and STING KO MEFs transfected with the indicated siRNAs. Immunoblot analysis of LC3-II conversion and p62 degradation in MEFs and ATG5 KO MEFs transfected with the siRNAs against STING. NC, Non-target control. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. Source data are available for this figure: SourceData F6.
Figure 6.

STING inhibits autophagosome – lysosome fusion. (A) Autophagic flux is accelerated in STING-KO MEFs compared with WT MEFs. WT MEFs and STING-KO MEFs were treated with Torin 1 with or without CQ for the indicated time and then immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (B) Inducible expression of STING at endogenous level inhibits autophagic flux in MEFs. Inducible stable cell line expressing STING was treated with different doses of doxycycline (0, 100, 200 ng/ml) for 24 h to induce STING expression. After 24 h, WT MEFs, STING-KO MEFs, and STING-KO MEFs reconstituted with inducible STING were treated with or without Torin 1 for 3 h. Cells were lysed and immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (C) Fusion between autophagosomes and lysosomes is promoted in STING-KO MEFs. WT MEFs, STING-KO MEFs, and STING-KO MEFs reconstituted with STING-HA were treated with Torin 1 for 3 h before fluorescence of GFP-RFP-LC3 was imaged. Scale bar, 5 µm. (D) Quantification of the results in C. Percentage of RFP + GFP-dots in total RFP dots. Data are mean ± SD for 100 cells. ***, P ≤ 0.001. (E) Expression of STING-HA inhibits the autophagic flux in HEK293T cells. HEK293T cells were transfected with empty vector, WT STING-HA. 24 h after transfection, cells were treated with Torin 1 combined with or without CQ for indicated time. Immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. (F and G) STING regulated autophagosome–lysosome fusion is STX17 (F), ULK1, ATG9, ATG14, and ATG5 (G) dependent. Immunoblot analysis of LC3-II conversion and p62 degradation in MEFs and STING KO MEFs transfected with the indicated siRNAs. Immunoblot analysis of LC3-II conversion and p62 degradation in MEFs and ATG5 KO MEFs transfected with the siRNAs against STING. NC, Non-target control. Normalized fold change of LC3II/Tubulin or p62/Tubulin was listed. Source data are available for this figure: SourceData F6.

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