Stocks of frozen viable cells were generated immediately after one or two passages, and low passage number cells were used for all experiments. protein expressions from total cell lysates (30?g protein per sample) were analyzed by Western blotting with JA9 and anti-acetyl-histone H3 antibodies. B: Relative protein expressions from a representative experiment. Densitometric values were normalized to the respective -actin loading control levels, and expressed as fold increase over the untreated controls in the case of each cell line. (TIF 990 kb) 12885_2018_4945_MOESM3_ESM.tif (991K) GUID:?FA89BFF2-1EEE-49D1-AA2F-FC40B0788A0F Additional file 4: Physique S3. 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 brought on by 2?M Ca2+ ionophore A23187, and fluorescent signal of the GCaMP2 Ca2+ sensor was followed by confocal imaging. F/F0 values represent individual cells (41 control and 59 E2-treated cells) collected from three impartial experiments. (TIF 602 kb) 12885_2018_4945_MOESM4_ESM.tif (602K) GUID:?1DFDC749-AA93-4F58-A618-977DF124DA8B Additional file 5: Physique S4. Effects of 17-estradiol (E2)??HDAC inhibitor treatments on PMCA4 protein expression in the ER- positive BT-474 and in the ER- unfavorable MDA-MB-231 breast malignancy cell lines. A: BT-474 and MDA-MB-231 cells were cultured in E2-free culture medium and treated with 1?nM E2??100?nM fulvestrant (fulv.)??4?mM VPA or 3?M SAHA for 4?days as indicated. Equal amounts (30?g) of total cell lysates were analyzed by Western blotting using the anti-PMCA4 (JA9), anti-ER- and anti-ER- antibodies. -actin served as a loading control. B: Relative PMCA4 protein expression in the examined cell lines. Densitometric values were normalized to the respective -actin levels and expressed as fold increase over untreated controls. Bars represent mean??SEM from three independent experiments. (TIF 915 kb) 12885_2018_4945_MOESM5_ESM.tif (916K) GUID:?1D7C8AE3-0066-4059-9E69-6FEF1BE46BEA Data Availability StatementThe datasets analyzed during the current study are available in the Oncomine database [35] and in the Cistrome [40] and GEO [42] databases. Abstract Background Remodeling of Ca2+ signaling is an important step in cancer progression, GZ-793A and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by genes) is usually common in tumors. Methods In this study PMCAs were examined in breast malignancy datasets and in a variety of breast malignancy cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. Results Three distinct datasets displayed significantly lower mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of and was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast malignancy cell lines revealed low PMCA4b expression in the ER- positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER- pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER- positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER- binding site in the gene in MCF-7 cells but not in other ER- positive tumor cells. In the triple unfavorable cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared.discussed that the ability of ER- to regulate gene expression in different cell lines in a different way depended on many factors, such as varying transcription factor expression and activity, different chromatin structure or epigenetic modifications [68]. after a 4?day VPA or SAHA treatment. Densitometric values were normalized to the respective -actin loading CCL4 control levels and expressed as fold increase over the untreated controls. Bars represent mean??SEM from two to four independent experiments. (TIF 5113 kb) 12885_2018_4945_MOESM2_ESM.tif (4.9M) GZ-793A GUID:?4EB990D2-96CA-4FB4-9CB9-AE723879B2A1 Additional file 3: Figure S2. Effects of VPA and SAHA treatments on PMCA4b protein expression and histone H3 acetylation level in different breast cancer cell lines. A: Cells were treated with 4?mM VPA or 3?M SAHA for 4?days, and protein expressions from total cell lysates (30?g protein per sample) were analyzed by Western blotting with JA9 and anti-acetyl-histone H3 antibodies. B: Relative protein expressions from a representative experiment. Densitometric values were normalized to the respective -actin loading control levels, and expressed as fold increase over the untreated controls in the case of each cell line. (TIF 990 kb) 12885_2018_4945_MOESM3_ESM.tif (991K) GUID:?FA89BFF2-1EEE-49D1-AA2F-FC40B0788A0F Additional file 4: Figure S3. 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?M Ca2+ ionophore A23187, and fluorescent signal of the GCaMP2 Ca2+ sensor was followed by confocal imaging. F/F0 values represent individual cells (41 control and 59 E2-treated cells) collected from three independent experiments. (TIF 602 kb) 12885_2018_4945_MOESM4_ESM.tif (602K) GUID:?1DFDC749-AA93-4F58-A618-977DF124DA8B Additional file 5: Figure S4. Effects of 17-estradiol (E2)??HDAC inhibitor treatments on PMCA4 protein expression in the ER- positive BT-474 and in the ER- negative MDA-MB-231 breast cancer cell lines. A: BT-474 and MDA-MB-231 cells were cultured in E2-free culture medium and treated with 1?nM E2??100?nM fulvestrant (fulv.)??4?mM VPA or 3?M SAHA for 4?days as indicated. Equal amounts (30?g) of total cell lysates were analyzed by Western blotting using the anti-PMCA4 (JA9), anti-ER- and anti-ER- antibodies. -actin served as a loading control. B: Relative PMCA4 protein expression in the examined cell lines. Densitometric values were normalized to the respective -actin levels and expressed as fold increase over untreated controls. Bars represent mean??SEM from three independent experiments. (TIF 915 kb) 12885_2018_4945_MOESM5_ESM.tif (916K) GUID:?1D7C8AE3-0066-4059-9E69-6FEF1BE46BEA Data Availability StatementThe datasets analyzed during the current study are available in the Oncomine database [35] and in the Cistrome [40] and GEO [42] databases. Abstract Background Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by genes) is common in tumors. Methods In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. Results Three distinct datasets displayed significantly lower mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of and was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER- positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER- pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER- positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER- binding site in the gene in MCF-7 cells but not in other ER- positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER- positive cells. Although, the expression of PMCA4b was relatively high in the triple negative GZ-793A cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. Conclusions Our results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression. Electronic supplementary material The online version of this article.