Fig. 2: Wild-type RUFY3, but not the Arl8b-binding-defective mutant, promotes perinuclear lysosome clustering.

a–c Confocal micrographs of HeLa cells expressing RUFY3-HA (WT) (a), RUFY3 (∆446–561)-HA (b), and RUFY3 (RK → A)-HA (c) and stained for lysosomes using an anti-LAMP1 antibody. Transfected cells are marked with a boundary. d A schematic depicting the quantification method employed for analyzing the distribution of LAMP1-positive compartments in a cell. e Quantification of the distribution of LAMP1-positive compartments in HeLa cells transfected with the indicated plasmids for the experiments shown in a–c. The values plotted are the mean ± SD from three independent experiments. The total number of cells analyzed is indicated on the graph (****p < 0.0001; n.s. not significant; two-tailed Student’s t-test). f–i Confocal micrographs of HeLa cells transfected with Arl8b-FLAG alone (f) or co-transfected with indicated RUFY3 expressing plasmids (g–i) and stained with indicated antibodies. The cell boundary is marked with a line and yellow arrows mark the peripheral localization of Arl8b-positive vesicles. j, k Colocalization analysis of Arl8b with indicated RUFY3 proteins was assessed by calculating Pearson’s correlation coefficient (j) and Mander’s overlap (k) from the experiments shown in g–i. 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 graph (****p < 0.0001; two-tailed Student’s t-test). Scale Bars: 10 µm (main); 2 µm (inset).