Fig. 4: Effect of RXFP2 and CASZ1 overexpression in H295R-S2 cells.
From: Identification of risk loci for primary aldosteronism in genome-wide association studies
a RXFP2 and CASZ1 mRNA expression in H295R-S2 cells stably transfected with expression vectors coding for pcDNA3 (Ctrl), RXFP2 (RXFP2), or CASZ1 (CASZ1). mRNA expression is normalized against the geometric mean of three housekeeping genes. RXFP2 vs Ctrl, ***p = 0.0002; CASZ1 vs Ctrl ****p < 0.0001; unpaired t test, two-sided. b Effect of RXFP2 and CASZ1 overexpression in stably transfected H295R-S2 cells on cell proliferation. Cells were seeded in 96-well plates at a density of 5000 cells/well and grown for 4 days. Basal and stimulated aldosterone (c) and cortisol (d) production by H295R-S2 cells stably expressing RXFP2 or CASZ1. H295R-S2 cells were serum deprived for 24 h and then incubated for another 24 h with fresh serum deprived medium in absence (basal) or presence of 10 nM AngII. Aldosterone and cortisol levels are indicated as fold induction over mock-transfected cells in basal conditions. Aldosterone: Ctrl+AngII vs Ctrl ****p < 0.0001; RXFP2 vs Ctrl ****p < 0.0001; CASZ1 vs Ctrl **p = 0.0013; RXFP2 + AngII vs Ctrl+AngII ****p < 0.0001; CASZ1 + AngII vs Ctrl+AngII **p = 0.0013. Cortisol: Ctrl+AngII vs Ctrl ****p < 0.0001; RXFP2 + AngII vs Ctrl+AngII ****p < 0.0001. e Representative Western blot of aldosterone synthase expression in H295R-S2 cells stably expressing RXFP2 and CASZ1. f Quantification of aldosterone synthase expression (using tubulin as a loading control). Values are represented as mean + SEM of two independent experiments performed in quaduplicate (in a, median + interquartile range of one experiment performed in triplicate). Ctrl+AngII vs Ctrl ****p < 0.0001; CASZ1 vs Ctrl **p = 0.0052; CASZ1 + AngII vs Ctrl+AngII **p = 0.0052. c, d, f: Global comparison was evaluated using ANOVA or Kruskall Wallis test followed by Sidak’s or Dunn’s multiple comparison test. Cells overexpressing RXFP2 or CASZ1 were analyzed separately. Comparison between Ctrl vs Ctrl+Ang was performed with two-sided unpaired t test to control for cell response to AngII.
