Fig. 5.

Deletion of TRPML1 enlarges osteoclastic lysosome size and impairs bone resorption. (A) BMMs isolated from WT and TRPML1^−/− mice were cultured with and without RANKL for 2 days, as indicated. The lysosomes were then loaded with Lysotracker and imaged by confocal microscopy by random selection of images. Lysosome sizes were then analyzed based on lysosome circumference. The figure shows representative images (Scale bar = 10 μm) and lysosome size under each condition. The results are from 3 experiments and 4 images were randomly chosen from each experiment. (B) Effects of TRPML1 deficiency on the TRAP secretion was determined by its release into culture medium. The media were collected at the indicated times and used to measure TRAP activity. TRAP secretion was normalized to the activity of total TRAP collected by lysing the cells. Fold change in TRAP activities compared to WT without RANKL, is shown as the mean±SD of 3 experiments. *p < 0.05, **p < 0.01 respectively. (C) Bone-resorptive activity of WT and TRPML1^−/− osteoclasts was evaluated by seeding BMMs on hydroxyapatite-coated dishes and culturing for 6 days with or without RANKL. After removal of the cells, pits formed by mature osteoclasts were measured using ImageJ software. Images of RANKL-treated WT and TRPML1^−/− cells are shown. (Scale bar = 100 μm). Pit area relative to RANKL-treated WT cells is shown as the mean±SD of 3 experiments.