4.10. Treatments of CASR-HEK293A and PAds-Derived Cells with [Ca2+]o or R568

Before treatments, the complete culture medium was replaced with a serum starvation medium (serum-free medium supplemented with 0.2% BSA and 1% l-Glutamine), and cells were incubated overnight. Forty-eight hours after transfection, the CASR-HEK293A cells were pre-treated for 30 min with physiological saline solution (PSS) (NaCl 125 mM, KCl 4 mM, HEPES 20 mM, D-Glucose 0.1%, NaH₂PO₄ 0.8 mM, MgCl₂ 1 mM, pH 7.45), supplemented with 0.1% BSA. Subsequently, cells were treated in PSS with increasing concentrations of the calcimimetic R568 (Cayman Chemical Company; 50 nM, 100 nM, 0.5 μM, 5 μM, in the presence of 1.5 mM [Ca²⁺]_o). Treatments were carried out for 10 min, 60 min, or for 6 h to perform analysis of total protein, subcellular protein fractions, or gene expression, respectively. Untreated cells (NT) were used as controls. The experiments were also performed on cells transfected with the empty vector to verify the specificity of R568 effects on the CASR receptor. PAds-derived cells were treated with the CASR agonist R568 in the same experimental setting used for CASR-HEK293A cells for 60 min or 6 h to perform proteins and gene expression analysis, respectively.