Fig. 2: Ca²⁺ mobilizing agents induce TMEM175-independent lysosomal H⁺ leakage.

a Representative images of live HeLa-Lyso-mKeima cells treated with DMSO or ebastine (left) and corresponding mKeima FLIs (right). b Kinetics of mKeima FLI in HeLa cells treated with 6 μM terfenadine for 60 min. c Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 6 μM terfenadine for the last 20 or 40 min. d Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 15 μM ebastine (left) or 6 μM terfenadine (right) for the last 20, 40 or 60 min. e Relative Fluo-4-AM (left), mKeima (middle) and SypHer3 (right) FLIs in HeLa cells treated with 6 μM terfenadine, 500 nM thapsigargin, 10 μM ML-SA1 or 1 μM ATP for the last 5 min and analyzed by flow cytometry. See Supplementary Fig. 4g for gating of the cells for flow cytometry analyses. f Relative mKeima FLI in HeLa-Lyso-mKeima cells pretreated for 30 min with 20 μM BAPTA-AM and treated with 500 nM thapsigargin for the last 5 min. g Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 10 μM ML-SA1 (left) or 500 nM thapsigargin (right) for the last 5 min. Bars, SD of three independent experiments with 10 (a), 67 (b), 30 (c, d, f, g), or ≥10000 (e) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-tailed, homoscedastic t-test (a), one-way Anova with Tukey’s multiple comparisons (b) or two-way Anova with Dunnett’s multiple comparisons (c–g).