Fig. 1: Direct encapsulation of STING into the lumen of Lamp1-positive compartments. | Nature Cell Biology

Fig. 1: Direct encapsulation of STING into the lumen of Lamp1-positive compartments.

From: STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes

Fig. 1

a, Sting−/− MEFs stably expressing mRuby3-STING and Lamp1-EGFP were treated with DMXAA. For the inhibition of lysosomal proteolysis, E64d and pepstatin A were added to the medium. Cells were fixed and imaged. b, The fluorescence intensity of mRuby3-STING in a was quantified. c, Cells were stimulated with DMXAA in the presence of E64d/pepstatin A for the indicated times. Data are presented as the ratio (%) of [mRuby3-STING in Lamp1-positive areas (Lamp1+)]/[mRuby3-STING in whole cell]. d, (1) ‘Macroautophagy’; STING vesicles are first occluded into autophagosomes, which then fuse with lysosome. (2) ‘Membrane fusion’; STING vesicles fuse with endosome or lysosome, followed by invagination of limiting membrane of endosome or lysosome, yielding intraluminal STING vesicles. (3) ‘Encapsulation by endosome or lysosome’; STING vesicles are directly encapsulated into endosome or lysosomes. e–g, TfnR-EGFP and mRuby3-STING were stably expressed in Sting−/− MEFs. Cells were treated with DMXAA and then with LysoTracker Deep Red. The boxed area in the bottom panels is magnified in the top panels (e). Fluorescence intensity profile along the white line in e is shown (f). Cells were treated with DMXAA or HT-DNA and then with LysoTracker Deep Red. Data are presented as the ratio (%) of [TfnR-EGFP in LysoTracker-positive areas (LysoTracker+)]/[TfnR-EGFP in whole cell] (g). h, Sting−/− MEFs stably expressing mRuby3-STING, Lamp1-EGFP and mTagBFP2-Rab5 were treated with DMXAA. The white boxed area is magnified in the right panels. mTagBFP2-Rab5-positive area and Lamp1-EGFP-positive area are magnified at the bottom, respectively. The fluorescence intensity of mRuby3-STING within Rab5+ or Lamp1+ compartments was quantified. i, EGFP-Rab5 or Lamp1-EGFP was stably expressed in Sting−/− MEFs reconstituted with mRuby3-STING. Data are presented as the ratio (%) of [mRuby3-STING inside Rab5+ or Lamp1+]/[mRuby3-STING in whole cell]. NS, not significant. Scale bars, 5 µm (a), 10 µm (e,h) and 1 µm (magnified images in e and h). Data are presented in box-and-whisker plots with the minimum, maximum, sample median and first versus third quartiles (b,c,g–i). The sample size (n) represents the number of cells (b,c,g,i) or vesicles (h). Source numerical data are available in source data.

Source data

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