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Fig. 4 | BMC Cancer

Fig. 4

From: Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells

Fig. 4

PMCA4b localization and function is impaired in the triple negative MDA-MB-231 cell line. a: Cell culture morphology analysis of the MCF-7 and MDA-MB-231 cell lines. Pictures were taken with a phase contrast microscope. Gray cell masks were made using the ImageJ software v1.51j8 to emphasize the morphology of the cell cultures and cell-cell contacts. Scale bar: 30 μm. b: Subcellular localization of the PMCA4b protein in VPA-treated MCF-7 and MDA-MB-231 cells. MCF-7 cells were treated with 2 mM VPA, MDA-MB-231 cell were treated with 4 mM VPA for 4 days and immunostained with an anti-PMCA4b antibody (JA3). Images were taken by confocal microscopy. White arrows indicate PMCA4b protein in intracellular compartments. Scale bar: 30 μm. c: Relative PMCA4b expression in MCF-7 and MDA-MB-231 cells after a 4 day VPA treatment. Densitometric values of Western blots were normalized to the respective β-actin loading control levels and expressed as fold increase over the untreated controls. Bars represent mean ± SEM from two to four independent experiments. d: Ca2+ signal measurements in VPA-treated GCaMP2-MCF-7 and GCaMP2-MDA-MB-231 cells. Cells were treated with 4 mM VPA 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. F/F0 values represent individual cells (n = 14–32) from a representative experiment. e: Area under curve of the A23187-induced Ca2+ transients. Bar graphs are means ± SEM of the individual cells (n = 14–32). Significance between control and VPA-treated cells is denoted by *** (P < 0.001); two-tailed unpaired t-test

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