Fig. 4
Depletion of clathrin leads to hyperstabilization of HRS and TSG101 on endosomes. a Fluorescence intensity profiles over time. Representative from 11 (HRS/Clathrin LC) and 13 (CHMP4B/Clathrin LC) tracks in total from 5 (HRS/Clathrin LC) and 4 (CHMP4B/Clathrin LC) independent live-cell imaging experiments. b Frame-by-frame colocalization analysis of clathrin and HRS with EGF over time. See also Fig. 2b for details. Data from 8 independent experiments. c Western blot (WB) showing KD of endogenous clathrin HC (CHC). d Immunostaining reveals a hyperrecruitment of mCherry-HRS to endosomes depleted for clathrin HC in cells stimulated or non-stimulated with EGF. Total fluorescence intensities of endosomal mCherry-HRS per cell quantified by high-content microscopy and indicated as AU (×105) ± SD. Data from >2500 cells per condition from 4 independent experiments. Ctrl (−EGF) versus siCHC (−EGF): p < 0.001; Ctrl (+EGF) versus siCHC (+EGF): p < 0.001 (t-test). Arrows: mCherry-HRS and clathrin-positive EEA1 endosomes. e Immunostaining reveals a hyperrecruitment of endgenous HRS to endosomes depleted for clathrin HC. Quantification was done as in d. Data from >2500 cells per condition from 4 independent experiments. t-test: ***p < 0.001. Arrows: mCherry-HRS- and clathrin-positive EEA1 endosomes. f Live-cell imaging and fluorescence intensity profiles of tracked EGF-positive endosomes over time show a prolonged recruitment of mCherry-HRS to endosomes in clathrin HC-depleted cells. Representative examples of 12 (control) and 23 (siRNA clathrin HC) tracks from at least 4 independent experiments. g WB showing KD of endogenous clathrin HC (CHC). h Immunostaining reveals a hyperrecruitment of mEGFP-TSG101 to endosomes depleted for clathrin HC. Arrow: mEGFP-TSG101 localizing to a midbody. i Live-cell imaging and fluorescence intensity profiles of tracked EGF-positive endosomes over time show a prolonged recruitment of mEGFP-TSG101 and mCherry-HRS to endosomes in clathrin HC-depleted cells. Representative examples of 13 (control siRNA) and 15 (siRNA clathrin HC) tracks from at least 4 independent experiments. Scale bars, 20 µm and 5 µm (insets)
