In vitro cell proliferation assays were performed as described previously [50 (link), 51 (link)]. Briefly, VSMCs (2 × 103/well) were seeded into 96-well plates (Corning, Lowell, MA, USA). Cells were cultured in NG, Mtol and HG media supplemented with quercetagetin (5.5 μmol/L, Pim-1 inhibitor, Merck, Germany), stattic (10 μmol/L, STAT3 inhibitor, Merck) and dimethylsulphoxide (DMSO, ≤0.3%, v/v) for 48 hours. After washing twice with 1×PBS, a total of 100 μL DMEM plus 10 μL CCK-8 reagents (Beyotime Institute of Biotechnology, Jiangsu, China) were added to each well and the optical density (OD) was measured 4 hours later using a microplate reader (Multiskan MKS, Thermo Scientific, MA, USA) at dual wavelength (450/630 nm). Each group was duplicated in six wells and each assay was performed in triplicate.
Cell number was determined as described previously [52 (link)]. Briefly, VSMCs were seeded into 6-well plates (5 × 105 per well). After overnight recovery in an incubator at 37°C/5% CO2, the cells were serum-deprived for 12 hours and exposed to the aforementioned conditions for 48 hours and then counted using a hemocytometer ( n = 3 wells / group).
Cell number was determined as described previously [52 (link)]. Briefly, VSMCs were seeded into 6-well plates (5 × 105 per well). After overnight recovery in an incubator at 37°C/5% CO2, the cells were serum-deprived for 12 hours and exposed to the aforementioned conditions for 48 hours and then counted using a hemocytometer ( n = 3 wells / group).
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