Fig. 5: TMEM165 protects cells against cytosolic Ca²⁺ overload.

Cytosolic calcium concentrations analyzed by Fluo-4-AM in WT and TMEM165-KO MDA-MB-468 clones treated with 10 μM ML-SA1 (a) or 500 nM thapsigargin (b) for 0–300 sec or with 15 μM ebastine for 1 h (c). Death of indicated MDA-MB-468 cell clones treated with indicated concentrations of thapsigargin (d), ebastine (e, left) or terfenadine (e, right) for 24 h. Cells were stained with propidium iodide (dead cells) and Hoechst-33342 (total cells) and cell death was analyzed by Celigo Imaging Cytometer. f Cytosolic [Ca²⁺] in WT and TMEM165-KO HeLa clones treated with DMSO or 15 µM ebastine for 1 h and analyzed by Fluo-4-AM. g Death of indicated HeLa clones treated with 0–15 µM ebastine for 24 h and analyzed as in (d). h Cytosolic [Ca²⁺] in indicated HeLa clones treated with DMSO or 15 µM ebastine for 1 h and analyzed by Fluo-4-AM. i Death of indicated HeLa cell clones treated with 6 µM terfenadine or 15 µM ebastine for 24 h. Cells were stained with SYTOX Green (dead cells) and Hoechst-33342 (total cells) and cell death was analyzed by Celigo Imaging Cytometer. j Galectin 1 puncta (leaky lysosomes) in indicated HeLa clones treated with DMSO or 15 μM ebastine for the last 16 h. Bars, SD of three independent experiments with 30 (a, b), ≥5000 (c, f, h) or ≥15000 (d, e, i) cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-way Anova with Dunnett’s multiple comparisons.