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Depletion of TECPR2 leads to reduced localization of WASH complex subunits on early endosomes. A HeLa cells were treated with the indicated siRNA and incubated with ferrofluid (FF) for 30 min at 37 °C. The FF-containing compartments were purified and immunoblotted (IB) for the presence of indicated proteins. A non-specific band detected by the anti-FAM21C antibody is marked with an asterisk (*). B Densitometric analysis of WASH subunits upon TECPR2 depletion, normalized to the input and control siRNA. The data shown is mean±SD from three independent experiments, as shown in panel (A). Experiments are color-coded, where each dot represents the mean value (****p < 0.0001; ***p = 0.0009; *p = 0.0429; ns=0.9069; unpaired two-tailed Student’s t-test). C Representative confocal micrographs of HeLa cells treated with the indicated siRNA, followed by immunostaining with anti-FAM21C, -Strumpellin, and -WASHC1 antibodies. The inset shows the perinuclear localization of FAM21C in TECPR2-depleted cells. Scale bars: 10 µm (main); 5 µm (inset). D Quantification of the FAM21C, Strumpellin and WASHC1 puncta numbers in HeLa cells treated with the indicated siRNA. The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; ns=0.2885; unpaired two-tailed Student’s t-test). E Live-cell imaging of HeLa cells treated with indicated siRNA and expressing GFP-WASHC1 (green) and RFP-Rab5 (WT) (magenta) (see Supplementary Movie 11). Scale bars: 10 µm (main); 5 µm (inset). F Quantification of the Pearson’s colocalization coefficient (PCC) for GFP-WASHC1 with RFP-Rab5 (WT). The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). G Representative confocal micrographs of HeLa cells treated with the indicated siRNA, followed by immunostaining with anti-p16-Arc (green) and anti-Rab5 (yellow) antibodies and actin labeling using Alexa Fluor-647 conjugated phalloidin (magenta). Arrowheads in the insets indicate colocalized pixels. Scale bars: 10 µm (main); 5 µm (inset). H Quantification of the p16-Arc puncta number in HeLa cells treated with the indicated siRNA. The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). I Quantification of the PCC of p16-Arc and Rab5. The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). J Representative single frame from live-cell imaging movie of HeLa cells treated with indicated siRNA and expressing CapZβ-GFP (see Supplementary Movie 12). Scale bars: 10 µm (main); 5 µm (inset). K Quantification of CapZβ-GFP puncta in HeLa cells treated with control siRNA and TECPR2 siRNA. The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). Source data are provided as a Source Data file.
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