Fig 2: Fibroblast growth factor (FGF)-23 increases store-operated calcium channel expression in cardiac fibroblasts. Representative immunoblots and average data of phosphorylated-calcium/calmodulin-dependent protein kinase II (p-CaMKⅡ), calcium release-activated calcium channel protein 1 (Orai1), stromal interaction molecule 1 (STIM1) (A), and transient receptor potential canonical (TRPC) 1 channel, TRPC3 and TRPC6 (B) expression in control and FGF-23 (25 ng/mL)-treated cardiac fibroblasts (n = 5). * p < 0.05, ** p < 0.01.

Fig 3: NCS‐1 and ORAI1 silencing does not affect proliferation of GCaMP6m‐MDA‐MB‐231 cells nor promote the antiproliferative effect of doxorubicin. (A) Representative images of NT, NCS‐1, and ORAI1 siRNA‐transfected cells stained with EdU and Hoechst 33342 48 h after doxorubicin treatment (Scale bar = 50 µm). Graphs show the percentage of EdU‐positive cells in NCS‐1 and ORAI1 silenced cells 24 h (B, D) and 48 h (C, E) after doxorubicin treatment. Data shown represent mean ± SEM of four regions in duplicate wells from three independent experiments. Statistical analysis was performed using a repeated‐measures two‐way ANOVA with Bonferroni’s post‐hoc test.

Fig 4: NCS‐1 silencing suppresses unstimulated, basal Ca2+ influx which phenocopies ORAI1 silencing. (A) Relative mean [Ca2+]CYT increases in GCaMP6m‐MDA‐MB‐231 cells preincubated in PSS nominal and induced by the addition of extracellular Ca2+ [CaCl2 (1.8 mm) + BAPTA (100 µm)]. Graphs show analyses of (B) maximum relative [Ca2+]CYT increase and (C) rate of [Ca2+]CYT increase (from 9 to 100 s). (D–F) Confirmation of ORAI1 silencing using real‐time PCR and immunoblotting. (G, H) siRNA‐mediated inhibition of ORAI1 but not NCS‐1 suppresses SOCE, that is, Peak 2 during Ca2+ readdition at 750 s. Statistical analysis was performed using a one‐way ANOVA with Bonferroni’s post‐hoc test. ****P < 0.001 (I) Trace shows the Ca2+ readdition phase of wells not pretreated with CPA from the same experiment shown in (G) from 300 to 1800 s. The trace shows a reduction in unstimulated Ca2+ influx as a result of ORAI1 silencing. (J) Bar graph shows the mean ± SEM of the maximal [Ca2+]CYT increases during Ca2+ readdition from 700 to 1800 s. Statistical analysis was performed using a paired t‐test. *P < 0.05; **P < 0.002, ****P < 0.0001.

Fig 5: Expression profiles of calcium transporters in lactation.Mouse mammary tissue, starting from day 10 prior to parturition, was evaluated for the expression of (A) SPCA1, and (B) SPCA2 by Western blotting. Loading was normalized relative to tissue DNA concentrations, and expressed relative to starting levels at Day -10. C) RT-PCR of mRNA for isoforms of SPCA, Orai, PMCA and STIM proteins at distinct time points of mammary development, including pre-pregnancy (virgin), parturition (Day 0), lactation (Day 5) and involution (Day 5 after removal of pups). Transcripts are grouped as early, mid and unchanged, according to their time of induction following lactation on Day 0. D) SPCA2 was immunoprecipitated from lactating mouse mammary tissue using polyclonal rabbit anti-hSPCA2 peptide antibody or rabbit IgG as control. Orai1 was detected as a co-immunoprecipitate by immunoblotting (IB).