Fig. 7: RUFY3 depletion reduces lysosome size.

a pH calibration curve based on ratiometric fluorescence intensity measurements of Lysosensor Yellow/Blue DND-160. b Graph showing average pH value of lysosomes measured from HeLa cells treated with the indicated siRNAs. Values plotted are the mean ± SD from six independent experiments (n.s. not significant; two-tailed Student’s t-test). c, d Representative histogram showing mean fluorescence intensity (MFI) of Lysotracker Red DND-99 (LTR) uptake (1 h) in control siRNA- and RUFY3 siRNA-treated HeLa cells (c), and the graph in d represents the relative percentage of MFI for LTR uptake from three independent experiments (****p < 0.0001; two-tailed Student’s t-test). e, f Representative micrographs of live HeLa cells treated with the indicated siRNAs and labeled with LTR. The asterisk indicates cells transfected with the GFP-RUFY3 siRNA-resistant plasmid. Scale Bars: 10 µm. The quantification of the average fluorescence intensity of LTR-positive vesicles is shown in f. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and the total number of cells analyzed is indicated on the graph (****p < 0.0001; two-tailed Student’s t-test). g–j Representative TEM images of HeLa cells treated with the indicated siRNAs. Higher magnifications of lysosomes (dense and multi-lamellar structures, indicated by yellow arrowheads) are shown in the right panels. Scale Bars: 2 µm (main); 0.5 µm (inset). Lysosome size (i) and number (j) were quantified using TEM images. Note: in i, n represents the number of lysosomes analyzed for size measurement. k–n Representative SIM images of HeLa cells treated with indicated siRNAs and stained with anti-LAMP1 antibodies. Insets represent a magnified view of boxed areas, highlighting differences in lysosome size. The average area (m) and count (n) of LAMP1-positive vesicles per cell was measured in HeLa cells upon treatment with the indicated siRNAs. o–r Representative SIM images of HeLa cells treated with the indicated siRNAs and stained with anti-LAMP1 antibody. In the insets, zoomed views of selected ROIs are shown, and quantification of the average area of lysosomes is plotted (r). The values plotted are the mean ± SD and the total number of cells analyzed are indicated on the graph (****p < 0.0001; ***p = 0.0006; two-tailed Student’s t-test). Scale Bars: 10 µm (main); 1 µm (inset).