Arl8b is required for PLEKHM1 association with lysosomes and for its ability to promote clustering of LEs and lysosomes. (a) Control- and Arl8b-siRNA (#1 and #2)–treated HeLa cell lysates were immunoblotted (IB) with anti–Arl8 antibody for assessing the knockdown efficiency and α-tubulin as the loading control. The asterisk and arrowhead denote Arl8a and Arl8b protein bands, respectively. (b–e) Representative confocal micrographs depicting the localization of GFP-PLEKHM1 with dextran-647–loaded lysosomes in indicated siRNA treatments and Arl8b siRNA-rescued HeLa cells. Arrowheads mark colocalized pixels. (f–h) Representative confocal micrographs of HeLa cells treated with control- or Arl8b-siRNAs and transfected with GFP-PLEKHM1 followed by immunostaining for Rab7. Arrowheads mark colocalized pixels. (i) PC was calculated as a measure of colocalization of PLEKHM1 with dextran-647–loaded lysosomes or with Rab7 in control siRNA- and Arl8b siRNA-treated HeLa cells (n = 3; 30 cells analyzed per experiment). (j–m) Representative confocal micrographs of HeLa cells expressing GFP-PLEKHM1 and stained for LAMP1 in indicated siRNA treatments and Arl8b siRNA-rescued HeLa cells. Arrowheads mark colocalized pixels. (n) Mean size of PLEKHM1⁺ compartments in indicated siRNA treatments and Arl8b siRNA-rescued HeLa cells (n = 3; 10–18 cells analyzed per experiment). Data represent mean ± SEM (***, P < 0.001; ****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.