Fig. 3: Evidence of ‘direct encapsulation’ of STING by CLEM.
a–g, Sting−/− MEFs stably expressing mRuby3-STING (magenta) and Lamp1-EGFP (green) were treated with DMXAA in the presence of E64d/pepstatin A/orlistat (lipase inhibitor): Lamp1-positive endosomes/lysosomes and STING-positive vesicles (or structures) were identified by Airyscan super-resolution microscopy before processing for transmission EM to examine their ultrastructure (a); magnification of the boxed areas in a (b–e), with orange arrowheads in b and c indicating the membrane that surrounds STING vesicles (for EM images of serial sections, see Extended Data Fig. 5); a graphical image of lysosome containing membrane-encapsulated STING vesicles (f) (green arrowheads indicate limiting membrane of lysosome); the diameter of STING-positive membrane vesicles was measured and plotted as histograms (g). h, Sting−/− MEFs stably expressing mRuby3-STING (magenta), Lamp1-EGFP (cyan) and Halo-Rab11a (green) were treated with DMXAA for 6 h in the presence of E64d/pepstatin A/orlistat. i, The fluorescence intensity of Halo-Rab11a in lysosomes (Lamp1-positive areas) or in whole cell was quantified. Data are presented in box-and-whisker plots with the minimum, maximum, sample median and first versus third quartiles as the ratio (%) of [Halo-Rab11a in Lamp1+]/[Halo-Rab11a in whole cell]. j, Sting−/− MEFs stably expressing EGFP-STING (green) were treated with or without DMXAA. Cells were immunostained with anti-clathrin-heavy chain (CHC) antibody (magenta). k, TfnR-EGFP (green) and mRuby3-STING (magenta) were stably expressed in Sting−/− MEFs. Cells were treated with the indicated siRNAs, and then stimulated with DMXAA. Scale bars, 10 µm (a,j,k), 500 nm in (b–e,h) and 500 nm (magnified images in j and k). The sample size (n) represents the number of cells (i) or vesicles (g). Source numerical data are available in source data.
