Hy p0175

The rat thoracic aortic VSMCs were obtained from BeNa Culture Collection (BNCC340258, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Genview, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/mL) and streptomycin (100 U/mL). Cells were kept in a humidity incubator at 37 °C with 5% CO₂ in atmosphere. When 80% confluence in culture wells was reached, the cells were used in treatment.
In the follow-up experiments, the VSMC were divided into five groups: (a) Control group; (b) Ang-II group (VSMC were treated by 1 µM Ang-II); (c) Ang-II and metformin group [VSMC were treated by Ang-II (1 µM) and metformin (10 mM)]; (d) Ang-II, metformin, and LY294002 (HY-10108, MedChemExpress, China) group [VSMC were treated by 1 µM Ang-II, 10 mM metformin together with LY294002 (10 µM)]; and (e) Ang-II, metformin, and 740 Y-P (HY-P0175, MedChemExpress, China) group (VSMC were treated by 1 µM Ang-II, 10 mM metformin together with 740 Y-P (25 mM)).

An xCELLigence Real-Time Cell Analyzer (Acea Biosciences/Roche Applied Science) was used to assess barrier function of HUVEC monolayers. HUVEC 48 h post-transfection were seeded at a density of 60,000 cells/well of the E-plate (E-plate 16, Roche Applied Science), then electrical impedance readings acquired every 2 min for 24 h. Cells attached within the first 7-10 h and were fully confluent by 24 h. Results are reported at 24 h as the percent change in cell index calculated using the following formula: (Cell Index_siRNA−Cell Index_NT)/ABS(Cell Index_NT).
For drug treatments, HUVEC 48 h post-transfection were seeded to E-plates as above and grown to confluence for 24 h, then media was replaced with drugs and readings taken every 30 s. Drug concentrations were as follows: for contractility assays, 0.5 U/ml thrombin (Sigma-Aldrich, T7201-500UN) at 37°C for 15 min. For PI3K activation assays, 20 µM 740Y-P (MedChemExpress, HY-P0175) at 37°C for 1 h.

Confluent monolayers of HUVEC 48 h post-transfection were treated as follows. For contractility assays, HUVEC were treated with 0.5 U/ml thrombin (Sigma-Aldrich, T7201-500UN) at 37°C for 15 min. For contractility inhibition assays, HUVEC were treated with 10 µM blebbistatin (Sigma-Aldrich, B0560-1MG) at 37°C for 15 min. For PI3K activation assays, HUVEC were treated with 20 µM 740Y-P (MedChemExpress, HY-P0175) at 37°C for 22 h. For PI3K inhibition assays, HUVEC were treated with 100 nM wortmannin (SelleckChem, S2758) at 37°C for 22 h. For BMP9 ligand assays, HUVEC were serum starved in Endothelial Base Media (Lonza CC-3162) with 0.1% fetal bovine serum (FBS) for 24 h followed by treatment with 10 ng/ml BMP9 (R&D Systems, 3209-BP-010) at 37°C for 1 h. Immediately following drug treatments, HUVEC were fixed in warm 4% PFA at RT for 4 min.