Huvecs

Mouse brain microvascular endothelial cells (mBMECs, from Angio-Proteomie) were cultured in DMEM/HG medium (ThermoFisher, Waltham, MA, USA) supplemented with 20% FBS (ThermoFisher, Waltham, MA, USA), and 1% Antibiotic–Antimycotic (100×) solution (ThermoFisher, Waltham, MA, USA). Cells were infected with lentivirus carrying three short hairpin RNA (shRNA) sequences against mouse Sirt3 or a scramble lentivirus and selected with puromycin for stable expression. Puromycin was added 3 days after infection, with a concentration of 1 μg/mL, which was replaced every 2–4 days depending on the confluency of the cells. Human umbilical vein endothelial cells (HUVECs, from Lonza, Visp, Switzerland) between passages 6–9 were cultured in DMEM/F12 medium supplemented with 10% FBS, 1% Antibiotics–Antimycotics, and endothelial cell growth supplement (Sigma-Aldrich, St. Louis, MO, USA). HUVECs were transfected with human SIRT3 siRNA (GenePharma, Suzhou, China) or universal scrambled negative control siRNA using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher, Waltham, MA, USA), according to the manufacturer’s instructions and were used within 36–48 h after transfection.

HUVECs were purchased from ScienCell (Santiago, MN, United States; Lot Number 28433), and cultured in endothelial cell medium (ECM, ScienCell) supplemented with 1% endothelial cell growth supplement (ECGS, ScienCell) and 5% FBS. The culture surfaces were pre-coated by fibronectin (ScienCell) which was diluted in PBS (1:100). Cells from the fourth to sixth generations were used for the experiments. At 80%–90% confluence, HUVECs were transfected with control, Gsα, or CREB siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). According to the manufacturer’s instructions, the culture medium was replaced with Opti-MEM (Gibco) and the RNA-lipid complexes were added to cells followed by incubating for 6 h at 37°C and replacing with normal medium. After 48 h, cells were collected for analysis. For the virus-mediated gene transduction, HUVECs were infected with adenovirus-expressing Gsα or lentivirus-expressing CREB (Vigenebio, Jinan, China) for 48 h followed by analysis. H89 and forskolin were purchased from Abcam.

Human umbilical vein cells (HUVECs, PromoCell, C-12208) were cultured in endothelial cell growth medium (PromoCell, C-22010). To knockdown the expression of PTBP1, HUVECs were infected with lentivirus-shRNA-PTBP1 (LV-sh-PTBP1) (Gene Pharma, Inc.) at a multiplicity of infection (MOI) of 50 at 37 °C for 24 h, and then cultured with fresh medium for another 48 h at 37 °C in a 5% CO2 incubator before subsequent experiments. The cells infected with control lentivirus-shRNA (Gene Pharma, Inc.; MOI, 50) were used as a negative control. For the migration assay, HUVECs were incubated with 10 μg/mL mitomycin C (MedChemExpress) at 37 °C in a 5% CO2 atmosphere for 2 h^{50 (link)}. The LV-sh-PTBP1 sequences were as follows: 5’-CGGCACAGTGTTGAAGATCAT-3’. For the overexpression of ARRB1-L or ARRB1-S, HUVECs were infected with ARRB1-L overexpression lentivirus or ARRB1-S overexpression lentivirus. At 72 h after infection, the growth area was scratched. The cells were then photographed in five different fields at 0 h and 20 h after scratching using a Leica DMi8 microscope, respectively. Cell migration ability was calculated as a percentage of the wound area difference between 0 h and 20 h to the wound area at 0 h by ImageJ software.