Effect of STING mutations on autophagy regulation. (A) MEFs stably expressing STING-HA and Flag-STX17 were treated with Torin 1 for 2 and 4 h or HT-DNA for 3 and 5 h, respectively, then the cells were lysed for immunoprecipitation and immunoblotting with indicated antibodies. (B) STING R232A and STING S366A mutants disrupt STX17–SNAP29–VAMP8 SNAREpin assembly via their interaction with STX17. HEK293T cells were transfected with empty vector, STING-HA WT, STING-HA R232A, STING-HA S366A with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (C) Enhanced interaction of STX17 with STING N154S and STING V155M further disrupts STX17–SNAP29–VAMP8 SNAREpin assembly. HEK293T cells were transfected with empty vector, STING-HA WT, STING-HA N153S, or STING-HA V155M with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody or anti-HA antibody. Immunoblotting was performed with the indicated antibodies. (D–F) Complementation of STING-KO MEFs with STING corrects their autophagy phenotypes. STING-KO and STING-KO MEFs complemented with HA-tagged STING WT (D), R223A mutant (E), or N154S mutant (F) were treated with Torin 1 with or without CQ for the indicated time. Immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II Tubulin or p62/Tubulin was listed. (G) STING C148A mutant is defective for STX17 binding. HEK293T cells were transfected with empty vector, STING-HA WT, or STING-HA C148A with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (H) STING-KO MEFs stably expressing STING-HA WT or STING-HA C148A mutant were treated with Torin 1 combined with or without CQ for the indicated time. Immunoblotting was performed with the indicated antibodies. (I) Quantification of the results in Fig. S4 E. Number of LC3 dots per cell. Data are mean ± SD for 100 cells. * indicates the comparison with STING WT, P ≤ 0.001. # indicates the comparison with STING KO, P ≤ 0.001. (J) Quantification of the results in Fig. S5. Percentage of LC3 dots with STX17. Data are mean ± SD for 100 cells. * indicates the comparison with STING WT, P ≤ 0.001. # indicates the comparison with STING KO, P ≤ 0.001. (K) Quantification of the results in Fig. S6. Quantitative analysis of acidified autophagosomes (GFP−RFP+) versus neutral autophagosomes (GFP+RFP+) per cell. Data are mean ± SD for 100 cells. * indicates the comparison of RFP number with STING WT, P ≤ 0.001. # indicates the comparison of RFP number with STING KO, P ≤ 0.001. Source data are available for this figure: SourceData F9.
Figure 9.

Effect of STING mutations on autophagy regulation. (A) MEFs stably expressing STING-HA and Flag-STX17 were treated with Torin 1 for 2 and 4 h or HT-DNA for 3 and 5 h, respectively, then the cells were lysed for immunoprecipitation and immunoblotting with indicated antibodies. (B) STING R232A and STING S366A mutants disrupt STX17–SNAP29–VAMP8 SNAREpin assembly via their interaction with STX17. HEK293T cells were transfected with empty vector, STING-HA WT, STING-HA R232A, STING-HA S366A with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (C) Enhanced interaction of STX17 with STING N154S and STING V155M further disrupts STX17–SNAP29–VAMP8 SNAREpin assembly. HEK293T cells were transfected with empty vector, STING-HA WT, STING-HA N153S, or STING-HA V155M with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody or anti-HA antibody. Immunoblotting was performed with the indicated antibodies. (D–F) Complementation of STING-KO MEFs with STING corrects their autophagy phenotypes. STING-KO and STING-KO MEFs complemented with HA-tagged STING WT (D), R223A mutant (E), or N154S mutant (F) were treated with Torin 1 with or without CQ for the indicated time. Immunoblotting was performed with indicated antibodies. Normalized fold change of LC3II Tubulin or p62/Tubulin was listed. (G) STING C148A mutant is defective for STX17 binding. HEK293T cells were transfected with empty vector, STING-HA WT, or STING-HA C148A with or without STX17-Flag. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (H) STING-KO MEFs stably expressing STING-HA WT or STING-HA C148A mutant were treated with Torin 1 combined with or without CQ for the indicated time. Immunoblotting was performed with the indicated antibodies. (I) Quantification of the results in Fig. S4 E. Number of LC3 dots per cell. Data are mean ± SD for 100 cells. * indicates the comparison with STING WT, P ≤ 0.001. # indicates the comparison with STING KO, P ≤ 0.001. (J) Quantification of the results in Fig. S5. Percentage of LC3 dots with STX17. Data are mean ± SD for 100 cells. * indicates the comparison with STING WT, P ≤ 0.001. # indicates the comparison with STING KO, P ≤ 0.001. (K) Quantification of the results in Fig. S6. Quantitative analysis of acidified autophagosomes (GFP−RFP+) versus neutral autophagosomes (GFP+RFP+) per cell. Data are mean ± SD for 100 cells. * indicates the comparison of RFP number with STING WT, P ≤ 0.001. # indicates the comparison of RFP number with STING KO, P ≤ 0.001. Source data are available for this figure: SourceData F9.

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