Fig. 5

17β-estradiol enhances PMCA4b protein expression and activity in MCF-7 cells. 17β-estradiol (E2)-dependence of PMCA4b protein expression and activity. a: MCF-7 cells were cultured in DMEM or in E2-free DMEM and treated with 1 nM E2 ± 100 nM fulvestrant (fulv.) for 4 days as indicated. Equal amount (30 μg) of total cell lysates were analyzed by Western blotting with isoform specific antibodies: anti-PMCA1 (NR1), anti-PMCA4b (JA3), anti-SERCA2 (IID8) and anti-SERCA3 (PL/IM 430). β-actin served as a loading control. b: Relative protein expression of PMCA1, PMCA4b, SERCA2 and SERCA3 isoforms. Densitometric values were normalized to the respective β-actin levels and compared to the protein expression of untreated cells cultured in DMEM. Bars represent mean ± SEM from three independent experiments. Significance is denoted by * (P < 0.05) or ** (P < 0.01); two-tailed paired t-test. c: Ca²⁺ signal measurement in E2-treated GCaMP2-MCF-7 cells. Cells were cultured in E2-free DMEM and treated with 1 nM E2 for 4 days. Before the measurement, culture medium was replaced by HBSS supplemented with 2 mM Ca²⁺. Ca²⁺ influx was triggered by 2 μM Ca²⁺ ionophore A23187, and fluorescent signal of the GCaMP2 Ca²⁺ sensor was followed by confocal imaging. Data represent means of normalized values (F/F₀) ± 95% CI of 41 control and 59 E2-treated cells collected from three independent experiments. d: Area under curve of the A23187-induced Ca²⁺ transients. Bar graphs are means ± SEM of the individual cells taken from three independent experiments. Significance between control and E2-treated cells is denoted by *** (P < 0.001); two-tailed unpaired t-test. e: Half time of decay of the A23187-induced Ca²⁺ transients. Bar graphs are means ± SEM of the individual cells taken from three independent experiments. Significance between control and E2-treated cells is denoted by *** (P < 0.001); two-tailed unpaired t-test