Fig. 3
CCC depletion leads to WASH-dependent increase in endosomal F-actin. a Confocal imaging for F-actin and WASH immunofluorescence staining of HeLa WT and the indicated CRISPR/Cas9 knockout cell lines. Representative images of three independent experiments are shown. Scale bars 20 μm, for zoomed images 10 μm. b Quantification of normalized mean fluorescent intensity (MFI) of F-actin on WASH-positive vesicles from a. Results for individual cells are plotted, along with the mean and s.e.m. for each group (n = 62 cells for WT, 45 for CCDC93 KO, 48 for COMMD3 KO, 50 for VPS35L KO, 43 for VPS26C KO, 48 for FAM45A KO); ***P < 0.0001 (one-way ANOVA and Dunnett’s test to control). c Confocal imaging for cortactin and FAM21 immunofluorescence staining in the indicated cell lines. Representative images of two independent experiments are shown. Scale bars 20 μm, for zoomed images 5 μm. d Quantification of cortactin immunofluorescence on FAM21-positive vesicles from c. Results for individual cells are plotted, along with the mean and s.e.m. for each group (n = 51 for WT cells, 63 for CCDC93 KO, 56 for COMMD3 KO); ***P < 0.0001 (one-way ANOVA and Dunnett’s test to control). e Confocal imaging of F-actin, EEA1, and WASH immunofluorescence staining for the indicated cell lines. Representative images of two independent experiments are shown. Scale bars, 10 μm and 5 μm on zoomed images. f Quantitation of endosomal F-actin deposition on EEA1-positive endosomes in the indicated cell lines (n = 35 cells for HeLa WT, 36 for CCDC93 KO shcontrol, 37 for CCDC93 shWASH). The mean MFI and the s.e.m. for each group are plotted; ***P < 0.0001 (unpaired two-tailed t test to CCDC93 KO shcontrol)
