Fig. 8: RUFY3 regulates nutrient-dependent lysosome repositioning.

a–e Representative confocal micrographs (shown as grayscale inverted) of HeLa cells treated with control siRNA or RUFY3 siRNA and incubated in the indicated media for 4 h. Post-treatment, cells were fixed and stained using an anti-LAMP1 antibody. The distribution of lysosomes based on the LAMP1 signal from these experiments is shown in e, and the values plotted are the mean ± SD from three independent experiments, and the total number of cells analyzed is indicated on the graph (****p < 0.0001; two-tailed Student’s t-test). f, g HeLa cells transfected with indicated siRNAs were grown in complete media or subjected to 2 h starvation using EBSS media in the absence or presence of Bafilomycin A1 (BafA1). Lysates from these cell types were IB with the indicated antibodies. Protein densitometric analysis of LC3B-II levels normalized to β-tubulin is shown in g. h, i Representative confocal images of control (h) and RUFY3-depleted (i) HeLa cells incubated in media lacking serum for 1 h in the presence of BafA1. Post-treatment, cells were fixed and stained for LAMP1 and LC3. In the insets, selected peripheral (PP) and perinuclear (PN) regions of the cell are magnified to show colocalized pixels of LC3 with LAMP1 (denoted by yellow arrowheads). j Colocalization of LAMP1 with LC3 for the experiments performed in h and i was analyzed by measuring Pearson’s correlation coefficient. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and the total number of cells analyzed is on the graph (n.s. not significant; two-tailed Student’s t-test). k Schematic representation of the BODIPY FL-BSA uptake and de-quenching in lysosomes. l, m Representative histogram showing mean fluorescence intensity (MFI) of de-quenched BODIPY FL-BSA after 2 h of incubation in control siRNA- and RUFY3 siRNA-treated HeLa cells as analyzed by flow cytometry (l), and the bar graph in m represents the relative percentage of MFI signal for de-quenched BODIPY FL-BSA after 2 h of incubation in HeLa cells treated with control- or RUFY3 siRNA calculated from three independent experiments (***p < 0.001; two-tailed Student’s t-test). Scale Bars: 10 µm (main); 2 µm (inset).