Effects of disrupting lysosomes on cytosolic Ca ²⁺ in SH-SY5Y cells. (a) Schematic depicting permeabilization of lysosomes by GPN. (b) Effect of GPN (50 µM) and NH₄Cl (5 mM) on depolarization-evoked Ca²⁺ signals in the indicated cell type (mean ± SEM, n = 3–4 independent biological replicates). (c) Schematic depicting inhibition of the lysosomal ion channel TRPML1 by the small molecule inhibitor, ML-SI3. (d) Effect of ML-SI3 (10 µM) on depolarization-evoked Ca²⁺ signals in the indicated cell type (mean ± SEM, n = 2–4 independent biological replicates). (e) Summary data (mean ± SEM from two to four independent biological replicates) quantifying the peak change in the Ca²⁺ signals and the area under the curve from the indicated cell line and treatment. Each point represents the mean response from a cell population. ns, nonsignificant, **P < 0.01, ***P < 0.001 (one-way ANOVA, Tukey’s test). (f) Confocal micrographs of SH-SY5Y cells expressing GFP-tagged TPC2 and TPC2^PD expressed in SH-SY5Y cells (green). Cells were counterstained with an antibody to LAMP-1 (red). Merged images are shown to the right. Scale bar: 7 µm.