Cell cycle analysis and western blot of cell cycle protein

Cell cycle analysis was performed by using propidium iodide (PI)-staining flowcytometry. Cells (5×10⁴cells/well in 6-well plate) were seeded and treated with PGV-1 (2 and 4 μM) for 24 hours. On the next day, cells were harvested, washed with PBS, and fixed gently (drop by drop) in 70% cold ethanol. The cells were stained with the staining solution containing 1 mg/mL PI, 10 mg/mL RNase and 0.1% (v/v) Triton X-100 (Merck). Cells were incubated for 5 minutes in the dark room, transferred into a flowcytometric tube, and analyzed by BD Accuri C6 flowcytometry (BD Bioscience). The protein extract of the treated cells were subjected for western blotting with cyclin-B1 antibody (Cell Signalling #41355).