PLEKHM1 and SKIP play opposing roles in regulating lysosome positioning. (a–c) Representative confocal images of HeLa cells transfected with vector, FLAG-PLEKHM1, or FLAG-SKIP and immunostained for Arl8 and LAMP1. (d and e) Representative confocal images of HeLa cells immunostained for Arl8 and PLEKHM1 or SKIP. Only cut-mask image of the colocalized pixels eliminating background and individual pixels are shown on the right. (f) Perinuclear index of colocalized Arl8/PLEKHM1 or Arl8/SKIP pixels were calculated (n = 3; 15–20 cells analyzed per experiment). (g–k) Representative confocal images of HeLa cells treated with indicated siRNAs and stained for Arl8 and LAMP1. (l) PI of LAMP1⁺-compartments in HeLa cells transfected with indicated siRNAs and siRNA-resistant constructs (n = 3; 14–19 cells analyzed per experiment). (m–p) Representative confocal micrographs of HeLa cells treated with the indicated siRNAs followed by 2-h incubation in acetate Ringer’s solution, pH 6.9, and immunostained for LAMP1 to mark lysosomes. To mark the cell boundary, actin staining was performed using phalloidin and the nucleus was stained using DAPI. (q) Quantification of perinuclear index in HeLa cells treated with indicated siRNAs followed by 2-h incubation in acetate Ringer’s solution (n = 3; 10–18 cells analyzed per experiment). Data represent mean ± SEM (****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.