Figure 2.

Effects of rubiarbonone C on cell cycle progression and cell cycle regulatory proteins. (A) Effects of rubiarbonone C on cell cycle progression. Serum‐deprived VSMCs were incubated with rubiarbonone C (5–20 μM) for 24 h followed by 25 ng·mL⁻¹ PDGF BB treatment for 24 h. After DNA staining with PI, the DNA contents of individual nuclei were analysed by flow cytometry. Each value shown was derived from counting >10 000 events. The numbers of cells in the G₀/G₁, S and G₂/M phases are expressed as % of 10 000 events (n = 3). (B) PCNA expression was measured by confocal microscopy. After cells were fixed with 4% formaldehyde and membrane‐permeabilized using 0.25% Triton X‐100, immunofluorescence staining was performed using anti‐PCNA and anti‐TRITC antibodies. Nuclei were stained with DAPI. Representative immunoblots and immunofluorescence images are shown (scale bar = 20 μm) (n = 3). (C) Inhibition by rubiarbonone C of PDGF BB‐stimulated expression and activation of cell cycle regulatory proteins. Serum‐starved VSMCs were incubated with rubiarbonone C (5–20 μM) for 24 h, followed by 25 ng·mL⁻¹ PDGF BB treatment for another 24 h. Levels of CDK2, CDK4, cyclin E, cyclin D1 and PCNA and Rb phosphorylation were determined by Western blotting. The band densities were normalized to those of β‐actin signals. Means ± SEM (n = 5 for each experimental group). The gel images shown are representative of those obtained from five independent experiments. *P < 0.05 versus PDGF BB alone.