Fig. 4

TECPR2 localizes to and maintains the peripheral distribution of early and recycling endosomes.

A Representative confocal micrographs of HeLa cells treated with the indicated siRNA that were untransfected or transfected with a GFP-tagged TECPR2 (rescue construct) followed by immunostaining with anti-Rab5 and anti-EEA1 antibodies. Cells are highlighted with a white line to visualize the cell edges. Arrowheads in the insets denote the individual endosomes in the two channels. The line-scan analysis shows the fluorescence intensity of both channels along the yellow line drawn in the merge image. Scale bars: 10 µm (main); 5 µm (inset). B, C Quantification of average puncta size of EEA1 (B) and Rab5 (C) from experiments such as that shown in panel (A). 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). D Quantification of early endosome distribution represented as fractional distance of Rab5 and EEA1 vesicles from experiments such as that shown in panel (A). The thin horizontal dashed line indicates the interquartile range, and the thick dashed line indicates the median of the data. n denotes the total number of cells analyzed from three independent experiments (^****p < 0.0001; unpaired two-tailed Student’s t-test). E Representative confocal micrographs of HeLa cells treated with the indicated siRNA that were either left untransfected or transfected with a FLAG-tagged TECPR2 (rescue construct), followed by immunostaining with anti-TfR, anti-Rab11, and anti-FLAG antibodies. Arrowheads in the insets indicate Rab11-positive tubules. Scale bars: 10 µm (main); 5 µm (inset). F Quantification of the percentage of HeLa cells treated with the indicated siRNA in which Rab11-positive tubules were observed. A minimum of 93 and 75 cells were analyzed for control and TECPR2 siRNA-treated samples, respectively. The values are represented as described for Fig. 2F. The error bar represents mean ± SD from three independent experiments (^****p < 0.0001; unpaired two-tailed Student’s t-test). G Quantification of the recycling endosome distribution represented as fractional distance of Rab11 and TfR vesicles from experiments such as that shown in panel (E). The values are represented as described for Fig. 4D, and n denotes the total number of cells analyzed from three independent experiments (^****p < 0.0001; unpaired two-tailed Student’s t-test). H Representative confocal images of HeLa cells treated with the indicated siRNA and immunostained with anti-SNX1 and anti-HRS antibodies. Arrowheads in the insets denote co-localized pixels. Scale bars: 10 µm (main); 5 µm (inset). I Quantification of the Pearson’s colocalization coefficient of SNX1 and HRS in HeLa cells treated with 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). Source data are provided as a Source Data file.