Figure 5.
Depletion of RUFY1 leads to enlarged lysosomes with features suggestive of lysosome dysfunction. (A–C) Representative confocal micrographs of HeLa cells treated with the indicated siRNA, then immunostained with anti-LAMP1 antibody. (D) Representative confocal micrograph of HeLa cells treated with RUFY1 siRNA #1 and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with anti-LAMP1 antibody. Bars: (main) 10 µm; (insets) 2 µm. (E) Quantification of the average area of the lysosomes per cell (LAMP1-positive compartments) upon indicated siRNA treatments. The graph shows the average area of lysosomes per cell. The values plotted are the mean ± SD from three independent experiments. In each experiment, 20–30 cells were analyzed (****P < 0.0001; n.s., not significant; unpaired two-tailed t test). (F) Electron micrographs of ultrathin cryosections of HeLa cells treated with either control or RUFY1 siRNA. Cells were fixed and immunogold labeled for endogenous LAMP1 (10 nm gold). Bars: 200 nm. (G) Quantification of the lysosome area (LAMP1-positive compartments) from electron micrographs of HeLa cells treated with the indicated siRNA. The error bars represent the mean ± SD of 95 compartments per condition (****P < 0.0001; unpaired two-tailed t test). (H) Ratiometric measurement of Lysosensor Yellow/Blue DND-160 dye fluorescence in HeLa cells treated with either control or RUFY1 siRNA to assess change in the pH of lysosomes. The data plotted are the mean ± SD from three independent experiments (n.s., not significant; unpaired two-tailed t test). (I) Serum-starved control siRNA or RUFY1-siRNA-treated HeLa cells were pulsed with EGF (100 ng/ml) for 10 min and chased in complete medium for 15, 30, 45 and 60 min. EGFR degradation was evaluated from immunofluorescence images by normalizing the residual mean EGFR fluorescence intensity at various chase times to the mean EGFR fluorescence intensity in the pulse-only sample. The values plotted are the mean ± SD from three independent experiments with 50–60 cells analyzed per time point in every experiment (****P < 0.0001; **P < 0.01; n.s., not significant; unpaired two-tailed t test). (J and K) Confocal images of HeLa cells treated with control or RUFY1 siRNA, followed by immunostaining with an anti-cathepsin D antibody. Bars: 10 µm. (L) Measurement of Correlated Total Cell Fluorescence (CTCF) values of the cathepsin D signal in HeLa cells treated with the indicated siRNA using ImageJ. Data represent mean ± SD from three independent experiments with 50 cells analyzed per experiment (*P < 0.05; unpaired two-tailed t test). (M and N) Confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with an anti-cathepsin D antibody. Bars: 10 µm. (O) Measurement of CTCF values of cathepsin D signal in HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct. Data represents mean ± SD from three independent experiments with 35–50 cells analyzed per experiment (n.s., not significant; unpaired two-tailed t test).

Depletion of RUFY1 leads to enlarged lysosomes with features suggestive of lysosome dysfunction. (A–C) Representative confocal micrographs of HeLa cells treated with the indicated siRNA, then immunostained with anti-LAMP1 antibody. (D) Representative confocal micrograph of HeLa cells treated with RUFY1 siRNA #1 and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with anti-LAMP1 antibody. Bars: (main) 10 µm; (insets) 2 µm. (E) Quantification of the average area of the lysosomes per cell (LAMP1-positive compartments) upon indicated siRNA treatments. The graph shows the average area of lysosomes per cell. The values plotted are the mean ± SD from three independent experiments. In each experiment, 20–30 cells were analyzed (****P < 0.0001; n.s., not significant; unpaired two-tailed t test). (F) Electron micrographs of ultrathin cryosections of HeLa cells treated with either control or RUFY1 siRNA. Cells were fixed and immunogold labeled for endogenous LAMP1 (10 nm gold). Bars: 200 nm. (G) Quantification of the lysosome area (LAMP1-positive compartments) from electron micrographs of HeLa cells treated with the indicated siRNA. The error bars represent the mean ± SD of 95 compartments per condition (****P < 0.0001; unpaired two-tailed t test). (H) Ratiometric measurement of Lysosensor Yellow/Blue DND-160 dye fluorescence in HeLa cells treated with either control or RUFY1 siRNA to assess change in the pH of lysosomes. The data plotted are the mean ± SD from three independent experiments (n.s., not significant; unpaired two-tailed t test). (I) Serum-starved control siRNA or RUFY1-siRNA-treated HeLa cells were pulsed with EGF (100 ng/ml) for 10 min and chased in complete medium for 15, 30, 45 and 60 min. EGFR degradation was evaluated from immunofluorescence images by normalizing the residual mean EGFR fluorescence intensity at various chase times to the mean EGFR fluorescence intensity in the pulse-only sample. The values plotted are the mean ± SD from three independent experiments with 50–60 cells analyzed per time point in every experiment (****P < 0.0001; **P < 0.01; n.s., not significant; unpaired two-tailed t test). (J and K) Confocal images of HeLa cells treated with control or RUFY1 siRNA, followed by immunostaining with an anti-cathepsin D antibody. Bars: 10 µm. (L) Measurement of Correlated Total Cell Fluorescence (CTCF) values of the cathepsin D signal in HeLa cells treated with the indicated siRNA using ImageJ. Data represent mean ± SD from three independent experiments with 50 cells analyzed per experiment (*P < 0.05; unpaired two-tailed t test). (M and N) Confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with an anti-cathepsin D antibody. Bars: 10 µm. (O) Measurement of CTCF values of cathepsin D signal in HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct. Data represents mean ± SD from three independent experiments with 35–50 cells analyzed per experiment (n.s., not significant; unpaired two-tailed t test).

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