Figure 4: MICU1-mediated Ca²⁺ buffering in OvCa cells evaluated using cisplatin.

(a) CP20 cells loaded with the fluorescent dyes Rhod-2AM (5 μM), 10 μM cisplatin caused rapid increase in [Ca²⁺]_m. (b,c) Silencing of MICU1 by siRNA increased [Ca²⁺]_m responses to cisplatin in CP20 (B) or OV90 (c) cells loaded with Rhod-2AM (5 μM) and values are mean±s.d. (d) Normalized GCaMP2-mt fluorescence in shRNA transfected (shCTL-OV90) or MICU1-specific shRNA transfected (shMICU1-OV90) cells stimulated with cisplatin (10 μM). The experiment was repeated three times (total no. of cells cell quantified: shCTL-OV90 n=158 and shMICU1-OV90 n=128). (e) Changes in [Ca²⁺]_m in OSE cells expressing Flag-MICU1 upon 10 μM cisplatin treatment as compared to empty vector (EV) transfected cells and values are mean±s.d. (f) Normalized GCaMP2-mt fluorescence in FTE188 cells transfected with empty vector (FTE+EV) or Flag-MICU1 (FTE+Flag-MICU1) cells stimulated with cisplatin (10 μM). The experiment was repeated three times (total no. of cells quantified: FTE+EV n=135 and FTE+Flag MICU1 n=433). (g) Cisplatin causes similar release of [Ca²⁺]_ER in presence or absence of MICU1 as determined in OV90 cells loaded with [Ca²⁺]_ER indicator dye Fluo-5N (5 μM) and values are mean±s.d. (h) Disruption of [Ca²⁺]_ER release by Pyr6 (50 nM) prevents cisplatin-induced changes in [Ca²⁺]_m of OV90 cells. Cells were pretreated with Pyr6 (50 nM) and Rhod-2AM (5 μM) for 15 min followed by 10 μM cisplatin treatment, fluorescence for Rhod-2AM was determined. Data are expressed as mean±s.d. *P<0.05 for comparisons among more than two groups, we performed ANOVA with Bonferroni’s correction. P<0.05 was considered statistically significant. All tests were two-sided. Horizontal line represents statistical comparison between respective groups. All the experiments were repeated independently at least three times and in triplicate.