Fig. 4

Enhancement of store-operated Ca²⁺ entry in chemoresistant IGROV1 sublines. a STIM1, STIM2, TRPC1, Orai1, Orai2, and Orai3 were detected using immunoblotting in IGROV1 parental (WT) and chemoresistant (CP, SRT) cells. β-actin served as the internal control. b Pre-incubation of IGROV1 parental (WT) and chemoresistant (CP) cells with 2 μM fura-2/AM at 37 °C for 30 min for cytosolic Ca²⁺ measurement using a single-cell fluorimeter. Depletion of ER lumen-resident Ca²⁺ was induced by treating cells in Ca²⁺-free buffer with 2 μM thapsigargin for 10 min. Representative tracings show the subsequent elevation of Ca²⁺ and indicated that SOCE occurred during the exchange of Ca²⁺-free buffer to 2 mM Ca²⁺ buffer for 5 min. The data in representative curves for the measurement of SOCE from three independent experiments (where, n ≥ 60 cells). c SOCE-mediated total Ca²⁺ elevation was calculated from area under the curve. a.u., arbitrary unit. d-e IGROV1-CP cells overexpressing EGFP-tagged paxillin were pretreated with 10 μM YM-58483 for 30 min. Time-lapse images were captured under a total internal reflection fluorescence microscope (TIRFM). Quantitative analysis of the (d) assembly and (e) disassembly rates of paxillin in IGROV1-CP cells. Each bar represents mean ± SEM from at least 20 cells. *: significant difference between chemoresistant (CP) and parental (WT) cells. **: p < 0.01; ***: p < 0.001 by Student’s t-test