Cell cycle analysis

VSMCs transfected with MCU siRNA or scramble or WT and MCU^−/− skin fibroblasts were grown to confluence and maintained in a post-confluent state for 48 h, and then split to 50% density. DMEM with 10% FBS was added for the indicated times (24 and 32 h for VSMCs and 16 h and 24 h for skin fibroblasts). For some experiments, treatment with Aphidicolin (2μg/ml) for 24 h was used to arrest skin fibroblasts at the G1/S cell cycle transition. After 24 h, cells were washed with PBS and cultured in DMEM with 10% FBS and PDGF (20 ng/mL) for 5 h. The cells were harvested, fixed with 75% ice cold ethanol, and stained with propidium iodide (stock solution 50 μg/mL) and analyzed in a LSRII Flow Cytometer (Becton Dickinson).