FUNDC1 promotes cytosol Ca2+ influx, and NFATC1 translocation and activation. (a,b) Representative and quantification of Fura-2 measurement of [Ca2+]cyt level in response to ATP (0.1 mM) in SKBR3 cells with and without FUNDC1 knockdown (n = 4). (c,d) Representative recordings and quantification of Ca2+ ER [Ca2+]ER level during store filling initiated by the addition of 100 nmol/L Ca2+ and 1.5 mM ATP in SKBR3 cells (n = 4). (e–h) [Ca2+] measurements in cytosol (e,f) and ER (g,h) by ATP stimulation in MCF-7 cells with and without FUNDC1 overexpression (n = 4). (i,j) Effect of the SOCE inhibitors (50 μM of ML9 or 2APB) treatment on [Ca2+]cyt level in response to ATP (0.1 mM) in MCF-7 cells with and without over-expression (OE) FUNDC1 (n = 4). (k) Western blot analysis of de-phosphorylation of NFATC1 by FUNDC1 overexpression. Value is the relative ratio of phosphorylation (p)-NFATC1 and total (t)-NFATC1 (n = 3). *P < 0.05, with OE-CN Ctrl. #P < 0.05, with OE-FUDNC1 Ctrl. (l) Representative immune-staining of subcellular localization of NFATC1 with FUNDC1 overexpression in MCF-7 cells after treatment with 50 μM ML9 or 2APB (n = 5). (m) NFATC1 activation in MCF-7 determined by transfection with an NFATC1-luciferase reporter showing stimulation by FUNDC1 overexpression, with rescued by pre-treatment with store-operated Ca2+ entry blockers ML9 and 2APB (n = 6). (n,o) Western blot assay shown that the regulation of three major SOCE members by FUNDC1 in SKBR3 and MCF-7 cells (n = 3). (p) STIM1 silence by siRNA cloud inhibit FUNDC1 over-expression induced NFATC1-luciferase activation in MCF-7 cells (n = 3). Data are mean ± SD. *P < 0.05.