The viability of HBVSMCs was measured with a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories) according to the manufacturer’s instructions. Briefly, HBVSMCs cells were seeded in 96-well plates (2 × 103 cells/well) and rendered quiescent by replacing the medium with 0.2% FBS for 24 h. Then the cells were treated with AngII with or without ClC-2 siRNA treatment according to the experimental design. The medium was replaced with fresh medium containing 10 μl of CCK-8 reagent followed by incubation for 4 h at 37 °C in a 5% CO2 atmosphere. The absorbance value was read at 450–540 nm using a microplate reader (Bio-Tek).
At the same time, cell proliferation was measured based on the incorporation of BrdU during DNA synthesis in proliferating cells. Cells were treated with 50 mM BrdU for 4 h at 37 °C and then fixed with 4% paraformaldehyde. After permeabilization with 0.4% Triton X-100 in 2% HCI for 15 min, the cells were incubated with BrdU antibody at 4 °C overnight followed by treatment with biotinylated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) for 1 h. The percentage of BrdU-positive cells was determined by counting the numbers of stained cells and total cells.
At the same time, cell proliferation was measured based on the incorporation of BrdU during DNA synthesis in proliferating cells. Cells were treated with 50 mM BrdU for 4 h at 37 °C and then fixed with 4% paraformaldehyde. After permeabilization with 0.4% Triton X-100 in 2% HCI for 15 min, the cells were incubated with BrdU antibody at 4 °C overnight followed by treatment with biotinylated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) for 1 h. The percentage of BrdU-positive cells was determined by counting the numbers of stained cells and total cells.