Cell Culture Conditions for VSMCs, A7r5, and HEK293T

hVSMCs (primary aortic smooth muscle cells, ATCC PCS-100-012), A7r5 cells (ATCC-CRL-1444) and HEK293T cells (ATCC-CRL-11268) were purchased from American Type Culture Collection. hVSMCs were cultured in vascular basal media (ATCC PCS-100-030) supplemented with the vascular smooth muscle growth kit (ATCC PCS-100-042), containing 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin. hVSMCs were used between passages 2–5. HEK293T and A7r5 cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium containing 10 mg ml⁻¹ sodium pyruvate, 2 mM l-glutamine, 10% (vol/vol) FBS, 1× non-essential amino acids, 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin. The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin 2–TEV fusion gene [27 (link)] were generously provided by the laboratory of Dr Bryan Roth and maintained in high-glucose Dulbecco's Modified Eagle's Medium supplemented with 10% (vol/vol) FBS, 1× non-essential amino acids, 100 U ml⁻¹ penicillin, 100 µg ml⁻¹ streptomycin, 50 µg ml⁻¹ hygromycin B and 2 µg ml⁻¹ puromycin. All cells were cultured in a humidified environment at 37°C, 5% CO₂.