Abnormal lysosomes in strumpellin HSP models. (A) HeLa cells were depleted of strumpellin using four individual siRNAs (strumpellin 1–4), fixed, and labeled for LAMP1, then the diameter of the largest lysosome in each cell was measured. The mean ± SEM percentage of cells with a largest lysosome >1.8 µm is shown (n = 6 repeats, 100 cells/repeat). The immunoblot verifies strumpellin depletion. (B) EM of endolysosomal structures in wild-type HeLa cells (mock) or HeLa cells subjected to strumpellin depletion using siRNAs 1–4. With each of the siRNAs, abnormal endolysosomal structures encompassing a range of appearances, from those containing very dense networks of membrane to looser coils of membrane, were observed (compare the two images shown for strumpellin siRNA 1). (C) HeLa cells were mock-transfected or depleted with strumpellin siRNA, then fixed and visualized for M6PR and LAMP1. Colocalization between the two markers was quantified and plotted in the corresponding histogram (n = 3 repeats, 15 cells per condition in each repeat). Bars: (EM) 500 nm; (confocal immunofluorescence microscopy [IF] main panels) 10 µm; (IF magnified insets) 5 µm. P-values generated by two-tailed Student’s t test.