Binding to Arl8b is necessary for PLEKHM1 function in regulating endocytic cargo trafficking to lysosomes. (a) Schematic illustrating the uptake and further processing of DQ-BSA, an endocytic cargo in the cells. (b–e) Representative confocal images of HeLa cells treated with indicated siRNAs and subjected to DQ-BSA uptake for 6 h. The cells were then fixed and analyzed for DQ-BSA fluorescence. (f) Measurement of fold change in the fluorescence intensity of DQ-BSA from 1h to 6 h (n = 3; 50 cells analyzed per experiment). (g–j) Representative confocal micrographs of HeLa cells treated with the indicated siRNAs and transfected with either siRNA-resistant PLEKHM1 (WT) or siRNA-resistant PLEKHM1 (HRR→A) construct and subjected to DQ-BSA uptake for 6 h. (k) Quantification of DQ-BSA trafficking in HeLa cells treated with indicated siRNAs and transfected with either siRNA-resistant PLEKHM1 (WT) or siRNA-resistant PLEKHM1 (HRR→A) construct (n = 3; 50 cells analyzed per experiment). (l) Western blot of mature cathepsin B and D levels in control- or PLEKHM1-siRNA–treated HeLa cells. (m) PC was measured for cathepsin D, and LAMP1 colocalization in control- or PLEKHM1-siRNA–treated HeLa cells (n = 3; 30 cells per experiment). (n) HeLa cells treated with control- or PLEKHM1-siRNA were incubated for 1 h in growth medium supplemented with cathepsin L substrate, and fluorescence intensity was measured by flow cytometry (n = 3; 10,000 cells analyzed per experiment). Data represent mean ± SEM (n.s., not significant; *, P < 0.05; **, P < 0.01; Student’s t test). Bars, 10 µm.