PLEKHM1 colocalizes with Rab7 and Arl8b and promotes perinuclear clustering of lysosomes. (a) Lysates from indicated siRNA treatments and from PLEKHM1 KO-HeLa cells were immunoblotted (IB) with anti-PLEKHM1 antibody for assessing the knockdown efficiency and α-tubulin as the loading control. (b and c) Immunofluorescence depicting the specificity of PLEKHM1 antibody in HeLa cells treated with control- and PLEKHM1-siRNA. (d–h) Representative confocal micrographs of HeLa cells showing endogenous staining of PLEKHM1 with different endocytic markers, and the Pearson’s correlation coefficient (PC) for PLEKHM1 is quantified (n = 3; 25–30 cells analyzed per experiment). (i–k) Representative confocal micrographs of HeLa cells transfected with Arl8b-HA alone or cotransfected with GFP-PLEKHM1 or -NΔ300 PLEKHM1, respectively, and stained for LAMP1. (l) Colocalization of WT and NΔ300 PLEKHM1 with Arl8b was assessed by measuring the PC (n = 3; 75 cells analyzed per experiment). (m) Quantification of perinuclear index of LAMP1⁺ compartments in HeLa cells transfected with indicated plasmids (n = 3; 15–18 cells analyzed per experiment). (n) Representative immunogold EM image of HeLa cells cotransfected with GFP-PLEKHM1 and Arl8b-HA and labeled with 10- and 15-nm gold particles, respectively. Boxed region is magnified on the right (Bar, 100 nm). Arrowheads mark colocalized pixels. Data represent mean ± SEM (n.s., not significant; **, P < 0.01; ****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.