Fig. 2: CADM1 variants increase CYP11B2 expression and aldosterone production.
a, Transfection of EV, WT, G379D and V380D CADM1. Fluorescent images of HEK293 cells transfected with pLOC EV, WT or mutant CADM1 during the production of lentiviruses for transduction of CADM1 into H295R cells. Transfected cells expressed tGFP (green) present in the pLOC vector. Cells overexpressing WT CADM1 appeared in clusters, whereas a more uniform, monolayer distribution of cells was observed in cells overexpressing mutant CADM1 or EV. Scale bar, 400 μm. b, Silencing of CADM1 by shRNA. Western blot of H295R cells transduced with EGFP-tagged shRNA either nontargeting or targeting CADM1 (shCADM1). Immunoblotting was performed with anti-CADM1 (Sigma-Aldrich, S4945) and anti-GAPDH antibodies on the same blot. Immunoblots showed a reduction in CADM1 protein in shCADM1 transduced cells compared to nontargeting transduced cells with similar total protein (estimated by GAPDH expression). The experiment was repeated once independently with similar results. Full-length blots are provided as Source Data Fig. 2. c–e, CADM1 mRNA expression (c), CYP11B2 mRNA expression (d) and aldosterone production (e), each in human adrenal cells overexpressing WT or mutant CADM1 or silenced for CADM1. Transduction of CADM1 (G379D or V380D) as its short (442 amino acids) or long (453 amino acids) isoform increased CYP11B2 (encoding aldosterone synthase) and aldosterone production. No effect was seen with overexpression of WT CADM1, whereas a decrease in CYP11B2 expression and aldosterone production was seen with silencing CADM1 (shCADM1). All values are expressed as fold change relative to EV or nontargeting control cells. Data are presented as mean values ±s.e.m. n = 3 independent wells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test for overexpression experiments. For CADM1 mRNA expression, F = 49.00, P < 0.0001; for CYP11B2, F = 158.4, P < 0.0001; for aldosterone, F = 149.0, P < 0.0001. Two-sided Student’s t-test was performed on silencing experiments. When compared to WT or shCADM1, *P = 0.0130, **P = 0.0029, ***P = 0.0003, ****P < 0.0001. NS, not significantly different between WT and mutant CADM1. The statistics used to produce these plots are provided as Source Data Fig. 2. UT, untransduced cells.
