STX17 and STING translocate to different types of autophagic vacuoles upon DNA stimulation. (A) LC3 is recruited to STING positive vesicles. MEFs stably expressing STING-GFP was treated with HT-DNA for 5 h and stained with GFP and LC3 antibody. Scale bar for large images, 5 µm; Scale bar for inset images, 1 µm. (B) STX17 translocates to STING negative but LC3 positive vesicles. MEFs stably expressing STING-GFP and mCherry-STX17 were treated with HT-DNA for 5 h and stained with GFP, mCherry, and LC3 antibody. Scale bar for large images, 5 µm; Scale bar for inset images, 1 µm. (C) mTOR inhibition negatively regulates DNA-induced IFNβ expression. WT cells were treated with Torin 1, HT-DNA, or HT-DNA plus Torin 1 for 5 h, and the indicated RNAs was extracted for qPCR. (D) STING mediates HT-DNA–induced autophagic response. WT and STING-KO MEFs were treated with HT-DNA or HT-DNA with Torin 1 for 3 or 8 h. Cells were lysed and analyzed for immunoblotting with indicated antibodies. (E) STX17 is not required for STING degradation. WT and STX17 knock out cells were treated with HT-DNA for 2 or 6 h. Cells were collected for immunoblotting with the indicated antibodies. (F) STING translocates to vesicles upon cGAMP stimulation. WT and STX17 knock out cells stably expressing STING-GFP were treated with cGAMP for 6 h and then fixed for immunostaining. Scale bar, 5 µm. Source data are available for this figure: SourceData F10.