Figure S4.
RUFY1 depleted cells show features of lysosome dysfunction and enhanced colocalization of CI-M6PR and Rab14. (A–C) Representative confocal images of HeLa cells treated for 60 h with the indicated siRNAs and incubated for 2 h with Lysotracker Red (LTR) before fixation. Bars: (main) 10 µm; (insets) 2 µm. (D) Measurement of the fold change in LTR intensity in HeLa cells treated with the indicated siRNAs and analyzed by flow cytometry. The values plotted are the mean ± SD from three independent experiments (**P < 0.01; *P < 0.05; unpaired two-tailed t test). (E–J) Confocal micrographs of HeLa cells treated with the indicated siRNAs followed by immunostaining with anti-Rab7 or anti-EEA1 antibodies. Bars: 10 µm. (K) HeLa cells were treated with the indicated siRNAs and subjected to BODIPY-BSA uptake for 7 h. The BODIPY-BSA fluorescence was analyzed by flow cytometry. The values plotted are the mean ± SD from three independent experiments (**P < 0.01; unpaired two-tailed t test). (L) Western blot analysis of pro-cathepsin D (Cat D), mature Cat D, and LAMP1 levels in either control or RUFY1 siRNA-treated HeLa cells. α-tubulin was used as a loading control. (M, N, P, and Q) Representative confocal images of HeLa cells treated with control or RUFY1 siRNA, followed by immunostaining with anti-CI-M6PR and anti-Rab14 antibodies (M and N) or anti-CI-M6PR and anti-Vps35 antibodies (P and Q). Bars: (main) 10 µm; (insets) 2 µm. (O and R) The Pearson’s colocalization coefficient of CI-M6PR with Rab14 (O) or CI-M6PR and Vps35 (R) was measured in HeLa cells treated with either control or RUFY1 siRNA. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; n.s., not significant; unpaired two-tailed t test). Source data are available for this figure: SourceData FS4.