Fig. 4.

STIM1 controls the activation of NFAT4. (A) Representative Western blot showing NFAT4-P and NFAT4-deP, respectively, in HASMCs transfected with shScramble, shSTIM1#1, or shSTIM1#5 and either unstimulated or stimulated for 5 or 15 min with 2 μM thapsigargin (Tg) in the presence of 2 mM Ca²⁺. (B) Quantification of percentage of NFAT4-deP to NFAT-P in shScramble (n = 3), shSTIM1#1 (n = 3), and shSTIM1#5 (n = 3) HASMCs from A using densitometry normalized to α-tubulin. (C) Representative images of shScramble, shSTIM1#1, and shSTIM1#5 HASMCs expressing NFAT4mCherry that are either unstimulated (time 0) or stimulated with 2 μM Tg in the presence of 2 mM Ca²⁺ for 15 min and 24 min. White arrowheads point to the cell nucleus. (Scale bar: 25 μm.) (D) Time-lapsed quantification of nuclear NFAT4mcherry translocation of shScramble (n = 41; black trace), shSTIM1#1 (n = 29; green trace), and shSTIM1#5 (n = 35; blue trace) HASMCs stimulated with 2 μM Tg in the presence of 2 mM Ca²⁺. Translocation is measured by dividing the fluorescence of a region of interest in the nucleus by that of a region of interest in the cytoplasm for each cell. Quantification of maximal (E) and slope (F) of NFAT4 translocation from C. (G) Using ELISA, IL-6 secretion is measured in shScramble (n = 4; gray circles), shSTIM1#1 (n = 4; green circles), shSTIM1#5 (n = 4; blue circles), and shScramble pretreated with 1 μM cyclosporine (n = 4; purple circles). HASMCs were stimulated overnight with either vehicle or 500 nM BK. For each condition, IL-6 secretion is normalized to total protein content in media. (H) Quantification of IL-6 in the BAL of saline-challenged Myh11^Cre (n = 10), HDM-challenged Myh11^Cre (n = 10), saline-challenged STIM1^smKO (n = 9), and HDM-challenged STIM1^smKO (n = 10) mice. *P < 0.05, **P < 0.01, ****P < 0.0001 when compared to shScramble or saline-challenged Myh11^Cre, #P < 0.05 when compared to HDM-challenged Myh11^Cre (one-way ANOVA with Dunnett’s test for multiple comparisons).