Figure 4.

CASR activation induces YAP1 signaling in primary PAd cultures. (a,b) YAP1 nuclear accumulation (a) or cytoplasmic abundance (b) were investigated in primary PAd cultures (n = 3) after stimulation for 1 h with the indicated concentrations of the CASR agonist R568 (* in panel a, p = 0.06; * in panel b, p = 0.018 in presence of 0.5 μM, and ** in panel b, p = 0.037 in presence of 5.0 μM); representative Western blots are shown with densitometric analyses (right panels). Bars, mean protein expression from three experiments relative to untreated cultures (NT) ± SEM. p-values are from one-way ANOVA. (c) Inhibition of the LATS1/2 protein levels in primary PAd-cultures (n = 3) upon treatment with the CASR agonist R568. Western blot and relative densitometric analysis (right panel) are shown. (d−f) Analysis by qPCR of the indicated YAP1-target genes expression in three primary PAds-derived cultures treated with R568 (* p = 0.037 in panel d; * p = 0.210 in panel e, and * p = 0.035 in panel f) by qPCR in 3 PAds-derived cell preparations. (g,h) The expression of the YAP1-targeted genes CYR61, CTGF, and WNT5A or of the key parathyroid genes PTH, GCM2, TBX1, CASR, and VDR was analyzed in primary PAds-cultures transiently silenced for YAP1 and treated with the CASR agonist R568. Data are expressed relative to untreated (black bars). Bars, mean gene expression from two experiments ±SEM.