(A) In HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), bath application of the ML1 channel agonist ML-SA1 (20 µM) in a low or 'zero' Ca2+ (free [Ca2+]<10 nM) external solution induced an increase in GCaMP3 fluorescence (F470). After washout for 5 min, repeated applications of ML-SA1 induced responses that were similar to or larger than the first one. Note that because baseline may drift during the entire course of the experiment (up to 20 min), we typically set F0 based on the value that is closest to the baseline. (B) The average Ca2+ responses of three ML-SA1 applications at intervals of 5 min (n=26 coverslips; Figure 1—source data 1). (C) Pre-treatment with lysosome-disrupting agent GPN for 30 min abolished the response to ML-SA1 in HEK-GCaMP3-ML1 cells. Washout of GPN resulted in a gradual re-appearance of ML-SA1 responses. See quantitation in Figure 1—figure supplement 2K. (D) Repeated applications of GPN resulted in Ca2+ release that was measured with the Ca2+-sensitive dye Fura-2 (F340/F380) in non-transfected HEK293T cells. (E) Application of Bafilomycin-A (Baf-A, 5 µM) and Concanamycin-A (Con-A, 1 µM) quickly (<5 min) abolished LysoTracker staining, an indicator of acidic compartments. (F) Acute application of Baf-A (5 µM) for 5 min did not block Ca2+ refilling of lysosomes in HEK-GCaMP3-ML1 cells. (G) Prolonged pre-treatment (3 hr) with Baf-A did not block Ca2+ refilling of lysosomes. (H) Quantification of 1st (p value = 0.11), 2nd (p=0.01), and 3rd (p=0.004) ML-SA1 responses upon Baf-A treatment (n=8) compared to control traces (n=6; Figure 1—source data 1). (I) Prolonged treatment (1 hr) with Con-A did not prevent lysosomes from refilling their Ca2+ stores. (J) Quantification of 1st (p=0.90), 2nd (p=0.33), and 3rd (p=0.66) ML-SA1 responses with Con-A pre-treatment (n=3; Figure 1—source data 1). (K) Con-A did not reveal differences in Ca2+ refilling responses to repeated applications of GPN in untransfected HEK293T cells. Panels A, C, D, F, G, I, and K are the average of 30–40 cells from one representative coverslip/experiment. The data in panels B, H, and J represent mean ± SEM from at least three independent experiments.