Fig. 5: ESCRT proteins are required for encapsulation of STING into the lumen of Lamp1-positive compartments.
a, TfnR-EGFP (cyan) and mRuby3-STING (magenta) were stably expressed in Sting−/− MEFs. Cells were treated with the indicated siRNAs, and then stimulated with DMXAA. Cells were immunostained with anti-pTBK1 (yellow) antibody. b, The Pearson’s correlation coefficient between mRuby3-STING and pTBK1, or between mRuby3-STING and TfnR-EGFP in a is shown. Data are presented in box-and-whisker plots with the minimum, maximum, sample median and first versus third quartiles. c–f, CLEM analysis of STING-positive vesicles. Sting−/− MEFs stably expressing mRuby3-STING (magenta) and Lamp1-EGFP (green) were treated with siRNA against Tsg101 (c,d) or Vps4a/b (e,f), and then stimulated with DMXAA. Lamp1-positive lysosomes and STING-positive membranes were identified by Airyscan super-resolution microscopy before processing for transmission EM to examine their ultrastructure (c,e). The yellow boxed areas in c and e are magnified in the right panels, respectively. The red boxed areas in EM images are magnified in the bottom right panels, respectively. STING-positive vesicles in c and e are indicated by magenta arrows. The diameters of STING-positive vesicles in Tsg101- or Vps4a/b-depleted cells were measured and plotted as histogram (d and f). Scale bars, 10 µm (a), 500 nm (magnified images in a), 1 µm (left CLEM images in c and e), 500 nm (fluorescence images in c and e), 100 nm (magnified EM images in c and e). The sample size (n) represents the number of cells (b) or vesicles (d,f). Source numerical data are available in source data.
