Fig. 3: Ca2+-induced lysosomal H+ leakage is mediated by TMEM165.
a Representative (n = 3) immunoblots of indicated proteins in HeLa cells treated with indicated siRNAs for 72 h. b Relative mKeima FLI 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. c Cytosolic pH analyzed by pHrodo-AM FLI in HeLa cells treated with indicated siRNAs for 72 h and with 15 μM ebastine or 6 μM terfenadine for 2 h. d Relative mKeima FLI 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 0–300 sec. e Perilysosomal pH analyzed by Lyso-mKeima FLI ratio (585/405 nm) in HeLa cells treated with indicated siRNAs for 72 h (left) or in TMEM165 overexpressed cells (right). f Representative (n = 3) immunoblots of indicated proteins in wild type (WT), TMEM165 overexpressed cell lines (top) and TMEM165-KO cell lines (bottom). g Lysosomal pH analyzed by LysoSensorTM Yellow/Blue FLI in HeLa TMEM165-KO cells (left) and TMEM165 overexpressed cells (right). h Cytosolic pH analyzed by pHrodo-AM FLI in HeLa TMEM165-KO cells (left) and TMEM165 overexpressed cells (right). i Representative (n = 3) immunoblots of indicated proteins in mCherry- and TMEM165-mCherry-trasfected HeLa clones. Bars, SD of three independent experiments with 30 (b–e, h) or ≥15000 (g) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparison (e-left, h-right), two-way Anova with Dunnett’s multiple comparison (b–d) or two-tailed, homoscedastic t-test (e-right, g, h-left).
