Extended Data Fig. 10: ESCRT-driven microautophagy terminates STING signaling.
a, Two cell lines [Sting−/− MEFs expressing mRuby3-STING, and Sting−/− MEFs expressing mRuby3-STING and siRNA resistant EGFP-Tsg101 (WT)] were mixed, treated with Tsg101 siRNA, and stimulated with DMXAA for 12 h. b, siRNA-resistant EGFP-Tsg101 (WT or ΔUEV) and mRuby3-STING were stably expressed in Sting−/− MEFs. Cells were treated with Tsg101 siRNA, and stimulated with DMXAA for 12 h. c, Sting−/− MEFs expressing mRuby3-STING were treated with the indicated siRNAs and stimulated with DMXAA for 12 h. Cells were then fixed, permeabilized, and immunostained with anti-Lamp1. Scale bars, 10 µm, 500 nm in the magnified images. d, qRT-PCR of the expression of Cxcl10 in MEFs that were treated with the indicated siRNAs followed by stimulation with DMXAA for 12 h. Data are presented as mean values +/− SD. e, Western blots of cell lysates of MEFs stimulated with DMXAA for the indicated times. The sample size (n) represents the number of the biological replicates (d). Source numerical data and unprocessed blots are available in source data. f, (Control cells) Active STING/TBK1 complex is encapsulated into the lumen of Lamp1-positive compartments by microautophgy. Thus the signalling is terminated. (ATP6v1b2- or Lamp2-depleted cells) The encapsulation of active STING/TBK1 complex proceeds (Extended Data Fig. 10c). Thus the signalling is terminated (Extended Data Fig. 10d). Please be noted that STING degradation is impaired because of the defect in the ability of lysosomal proteolysis. (ESCRT-depleted cells) The encapsulation is impaired, thus active STING/TBK1 complex remains in the cytosol, leading to the duration of the signalling. Recent studies indicate the involvement of C9orf72 (PMID: 32814898), BLOC1(PMID: 29033128), NPC1(PMID: 34290407) in the STING degradation. The exact site of action of these proteins in the membrane traffic that STING follows, namely, ‘ER-the Golgi-REs-lysosomes’ remains to be elucidated.
