Oxidative stress increases lysosomal levels of SEPT9, which promotes perinuclear clustering. (A) Confocal microscopy images (inverted monochrome) of COS-7 cells treated with NaAsO₂ (300 µM) for 30 min or deprived of glucose for 1 h and stained for LAMTOR4 (inverted green) and SEPT9 (inverted magenta). Scale bars, 20 µm. Insets show the outlined perinuclear regions in higher magnification. Scale bars, 5 µm. (B) Bar graph shows the percentage (mean ± SEM; unpaired t test) of total lysosomal area (LAMTOR4) that overlaps with endogenous SEPT9 per cell (n = 40–59) after NaAsO₂ treatment (300 µM, 30 min) or glucose deprivation (1 h). (C) COS-7 cells were transfected with plasmids that coexpress scramble control or SEPT9 shRNAs and GFP or shRNA-resistant SEPT9-GFP (wild type or T339G mutant). Images show lysosome (LAMP1) localization (green) and GFP fluorescence (magenta) after treatment with NaAsO₂ (300 µM) for 2 h. Arrows point to peripheral clusters of lysosomes. Scale bars, 20 µm. (D and E) Plots show the fraction (mean ± SEM; extra sum of squares F test) of total LAMP1 intensity across the distance of the cell radius, from the cell center to the periphery, after normalization for the length of each cell (n = 31–37). (F and G) HeLa cells were transfected with plasmids that coexpress scramble control or SEPT9 shRNAs and GFP or shRNA-resistant SEPT9-GFP (wild type or T339G mutant). Cells were stained with anti-LAMP1 after treatment with NaAsO₂ (400 µM) for 1 h. Plots show the fraction (mean ± SEM; extra sum of squares F test) of total LAMP1 intensity across the distance of the cell radius after normalization for the length of each cell (n = 55–64). ns, nonsignificant (P > 0.05); ***, P < 0.001.