Fig. 2.

RANKL-induced [Ca²⁺]i oscillations are attenuated by deletion of TRPML1 and inhibition of lysosomal Ca²⁺ storage without affecting ER Ca²⁺ content. (A, B) Bursting lysosomes with GPN (200 μM) and inhibition of lysosomal Ca²⁺ influx by treatment with bafilomycin A1 (100 nM) eliminate RANKL-induced Ca²⁺ oscillations in BMMs cultured with RANKL for 48 hours. (C) RANKL-induced [Ca²⁺]i oscillations were measured in BMMs isolated from WT and TRPML1^−/− mice and cultured for 48 hours in the presence of RANKL. Cells were continuously perfused with HEPES buffer throughout the experiments. The columns show the average frequency and amplitude of the Ca²⁺ oscillations. **p < 0.01. (D, E) ER Ca²⁺ content was evaluated in WT (black traces) and TRPML1^−/− BMMs (red traces) following 48 hours of incubation with (E) or without (D) RANKL (50 ng/mL). ER Ca²⁺ was measured by releasing it to the cytoplasm by exposing the BMMs to 10 μM of the SERCA pump inhibitor, CPA. Ca²⁺ influx mediated by store-operated Ca²⁺ channels was then evaluated by addition of 1 mM Ca²⁺ to the perfusate. The traces are the mean±SD of at least 3 experiments.