*4.5. Cell Culture and Transfection*

HAC15 adrenocortical cells were cultured as previously reported [33]. For experiments, cells were plated at a density of 4 × 105 cells/well in a 12-well plate for 48 h. After overnight incubation in low-serum medium (DMEM/F-12 containing 0.1% cosmic calf serum, and antibiotics), cells were stimulated with 100 nM AngII (reference value in normotensive individuals 24 ± 17 pM [34]) (Sigma #A9525) ± 1 μM irbesartan (Sigma #I2286) or forskolin (10 μM, Sigma #F6886) for 6, 12, and 24 h, and then harvested for RNA extraction, and gene-expression studies.

*CRY1* and *CRY2* gene silencing was performed using the Amaxa technology (Program X-005). One million cells were electroporated in 100 μL of Nucleofector solution R, using 2 μL of a 100-μmol/L solution of Silencer Select predesigned small interfering RNA (siRNA) (Thermo Fisher Scientific, Waltham, MA, USA). After electroporation, cells were plated in a six-well plate, and recovered for 24 h. The medium was then changed to experimental low-serum medium (0.1% cosmic calf serum), and the cells were starved overnight. The following morning, cells were harvested for RNA extraction, and gene-expression studies.
