Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: SPCA2, Kv10.1, Orai1 and DDR1 tissue expression. DDR1, Kv10.1, Orai1 and SPCA2 expression in breast cancer tissues. (A) Representative immunohistochemical staining of DDR1, Kv10.1, Orai1 and SPCA2 in serial sections of breast tissue. 40x magnifications of tumor (bottom line) is compared to matched normal (up line) tissues. Immunohistochemistry shows the expression of the different proteins in the same area of the sections; staining is higher in tumor areas. (B) Comparison of staining scores for DDR1, Kv10.1, Orai1 and SPCA2 in non-tumor vs tumor areas (n = 29; *** p < 0,001, Student T test for paired samples).

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: SPCA2 silencing counteracts collagen 1 resistance to apoptosis. (A) Effect of SPCA2 silencing on the apoptotic rate of MCF-7 cells. Cells were starved for 48 h and the apoptosis assay was carried out by annexin V/PI staining. (a) Dot blots of a representative experiment and (b) mean ± SEM of total apoptotic cells, N = 3, * p < 0.05, ANOVA followed by Holm-Sidak post hoc tests. (B) Effect of SPCA2, Kv10.1 and SPCA2 + Kv10.1 silencing (a) or SPCA2, Orai1 and SPCA2 + Orai1 silencing (b) on MCF-7 cell mortality. Cells were starved for 48 h and the mortality was measured by Trypan Blue assay; values are reported as mean ± SEM, N = 3, * p < 0.05, ANOVA followed by Holm-Sidak post hoc tests.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Apoptosis Assay, Staining

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: SPCA2, Kv10.1 and SPCA2 + Kv10.1 silencing inhibit the collagen 1-induced cell survival.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: Collagen 1 induced basal calcium entry rely on Orai1, Kv10.1 and SPCA2 activity. (A) Representatives traces of basal calcium entry imaging of MCF-7 cells after 48 hours of starvation treated (b) or not (a) with collagen 1. (B) Representatives traces of Manganese quench imaging of cells treated (b) or not (a) with collagen 1. Measurements were performed after cells were starved for 48 h (C) (a) Histograms representing the averages ± standard error of basal calcium entry (−Coll: n = 90 siCtl, n = 104 siSPCA2, n = 61 siKv10.1, n = 74 siOrai1; +Coll: n = 118 siCtl, n = 86 siSPCA2, n = 76 siKv10.1, n = 69 siOrai1, N = 3; * p < 0.05, ANOVA followed by Holm-Sidak post hoc tests). (b) Histograms representing the averages ± standard error of slope values obtained in Manganese quench experiences. (−Coll: n = 104 siCtl, n = 83 siSPCA2, n = 88 siKv10.1, n = 72 siOrai1, n = 91 siSPCA2-Kv10.1, n = 85 siSPCA2-Orai1; +Coll: n = 96 siCtl, n = 71 siSPCA2, n = 77 siKv10.1, n = 87 siOrai1, n = 94 siSPCA2-Kv10.1, n = 98 siSPCA2-Orai1, N = 3; * p < 0.05, ANOVA followed by Holm-Sidak post hoc tests). (D) Patch clamp recordings of basal calcium currents. (a) Whole cell currents recorded in MCF-7 cells after 48 hours of starvation treated with collagen I. Patch-clamp measurements were performed with specific bath solution to record basal calcium currents. 250 msec ramps from −100 mV to +100 mV from a holding potential of −40 mV were applied. Values are reported as mean ± SEM. (b) Histograms representing the averages ± standard error of currents values at −100 mV. siKV10.1 (n = 6); siCtl and siSPCA2-Kv10.1 (n = 5); siOrai1 and siSPCA2 (n = 4); siSPCA2-Orai1 (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, ANOVA followed by Tukey post hoc tests.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Activity Assay, Imaging, Patch Clamp

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: SPCA2 co-localizes and interacts with Kv10.1 and Orai1. (A) Effect of collagen 1 alone and with MβCD on SPCA2 interaction with Orai1 or Kv10.1. Representative western blot of Orai1 and Kv10.1 expression after immunoprecipitation with an anti-SPCA2 antibody. Results were normalized as a percentage of the untreated control condition (−Coll). Values are reported as means of all the experiments, N = 3. (B) Staining of (a) Orai1 (green) and SPCA2 (red) and their merge or (b) Kv10.1 (green) and SPCA2 (red) and their merge to visualize co-localization in MCF-7 cells treated or non-treated with collagen 1 after 48 h of starvation. (C) Histograms showing the means of the Manders Overlap Coefficient of co-localization of Orai1-SPCA2 and Kv10.1-SPCA2 in cells treated or not with collagen 1. (N = 2, n = 35; * p < 0.05, ** p < 0.01, Two samples t -test)

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Western Blot, Expressing, Immunoprecipitation, Staining

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: SPCA2 regulates Kv10.1 and Orai1 total and membrane expression. (A) Representative western blots (a) of Kv10.1 and Orai1 proteins levels in siCtl and siSPCA2 MCF-7 cells after 48 h of starvation treated or not with collagen 1. Results were normalized as a percentage of the untreated control condition (−Coll). Values are reported as means of all the experiments. (b) Histograms representing the averages ± standard error of results obtained by western blots. N = 5, ** p < 0.01 ; ANOVA followed by Holm-Sidak post hoc tests. (B) Effect of SPCA2 silencing on Kv10.1 and Orai1 membrane expression. Representative western blots showing membrane fraction enrichment of Kv10.1 (a) and Orai1 (b) proteins in siCtl and siSPCA2 MCF-7 cells after 48 h of starvation. The membrane fractions were obtained through a biotinylation protocol. Results were normalized as a percentage of the siCtl collagen 1 treated condition. Values are reported as mean of all the experiments. N = 3. (C) Whole cell currents (a) recorded in siCtl and siSPCA2 MCF-7 cells after 48 h of starvation treated with collagen 1. 500 msec voltage ramps from −100 to +100 mV from a holding potential of −40 mV were applied to record Kv10.1 channel activity. Values are reported as mean ± standard error. (b) Histograms representing the averages ± standard error of currents values at +100 mV. siCtl (n = 10), siOrai1 (n = 5); * p < 0.05; Two-samples t -test.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: Orai1 membrane fraction depends on calcium levels; Kv10.1 membrane fraction depends on calcium levels and Orai1. (A) Representative western blots showing membrane fraction of Kv10.1 (a) and Orai1 (b) proteins in MCF-7 cells after 48 h of starvation treated or not with EGTA 1.6 mM. The membrane extracts were obtained through a biotinylation protocol. Results were normalized as a percentage of control condition. Values are reported as mean of all the experiments. N = 3. (B) Representative western blot showing membrane fraction enrichment of Kv10.1 in siCtl and siOrai1 MCF-7 cells after 48 h of starvation. The membrane extracts were obtained through a biotinylation protocol. Results were normalized as a percentage of siCtl condition. Values are reported as mean of all the experiments. N = 3. (C) Whole cell currents (a) recorded in siOrai1 MCF-7 cells after 48 h of starvation treated with collagen 1. 500 msec voltage ramps from −100 to +100 mV from a holding potential of -40 mV were applied to record Kv10.1 channel activity. Values are reported as mean ± standard error. (b) Histograms representing the averages ± standard error of currents values at +100 mV. The siCtl values reported in gray came from the same recording as in Fig. . siOrai1 (n = 8); * p < 0.05; Two-samples t -test.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership

doi: 10.1038/s41598-018-37602-7

Figure Lengend Snippet: Collagen 1 stimulates Kv10.1 departure from Golgi through a SPCA2-dependent pathway. Confocal microscope images of MCF-7 cells infected with cell light Golgi-GFP vector at the beginning of the starvation and then immunostained with an anti-Kv10.1 antibody. The conditions analyzed were siCtl treated or not with collagen 1 and siSPCA2 treated with collagen 1. The white arrows indicate the co-localization between Kv10.1 and Golgi.

Article Snippet: The anti-Kv10.1 (Alomone Labs, Jerusalem, Israel) or anti-Orai 1 (ProSci Inc, CA, USA) antibodies were added at dilution of 1:100 for 1 hour and detected in a first step after a 3% NGS saturation with a biotinylated anti-goat secondary antibody (1:50, Jackson ImmunoReasearch Labs, Inc, PA, USA).

Techniques: Microscopy, Infection, Plasmid Preparation