Extended Data Fig. 1: Atg5-dependent macroautophagy was not involved in STING degradation.
a, Sting−/− MEFs stably expressing mRuby3-STING and Lamp1-EGFP were treated with DMXAA for 12 h. For the inhibition of lysosomal proteolysis, E64d and pepstatin A were added to the medium. The boxed areas in the top panels are magnified in the bottom panels. b, Fluorescence intensity profile along the white dotted line in (a) is shown. c, Cells were stimulated with DMXAA for the indicated times. The fluorescence intensity of mRuby3-STING in cells was quantified. d, Sting−/− MEFs stably expressing mRuby3-STING and Lamp1-EGFP were treated with HT-DNA for 12 h. The cells were then treated with DMXAA and protease inhibitors for 12 h. The boxed area is magnified in the right panel. e, The ratio (%) of [mRuby3-STING inside or outside Lamp1+]/[mRuby3-STING in whole cell] is indicated. f, Atg5 Tet-off cells were cultured with or without Doxycycline (Dox). Cells were stimulated with dsDNA for the indicated times. Cell lysates were analyzed by western blotting. g, mRuby3-STING and Lamp1-EGFP were stably expressed in Atg5 Tet-off cells. Cells were cultured with or without Dox for 4 days. The cells were then treated with DMXAA and protease inhibitors for 12 h. The boxed areas are magnified and shown. Fluorescence intensity profiles along the yellow lines are shown. h, The ratio (%) of [mRuby3-STING inside or outside Lamp1+]/[mRuby3-STING in whole cell] in (g) is indicated. Data are presented in box-and-whisker plots with the minimum, maximum, sample median, and first vs. third quartiles (c, e, and h). Scale bars, 10 µm in (a, d, and g), 500 nm in the magnified images in (a, d, and g). NS, not significant. The sample size (n) represents the number of cells (c, e, and h). Source numerical data and unprocessed blots are available in source data.
