Figure 2.

Comparison of butyrate and TSA effects on G1-specific cell cycle regulatory proteins. Proliferating VSMC were treated with different concentration of butyrate or TSA for 48 h (A and B) or treated with butyrate or TSA for indicated periods of time (C and D) to determine dose- and time-dependent effects of butyrate and TSA on G1-specific cell cycle proteins, respectively. At the end of treatment, cell lysates were prepared and processed for western analysis of cyclin D3, cdk4, cdk6, cdk2, p15INK4b, p21Cip1, Rb and phospho-Rb. Immunoblotting of ERK1/2 was performed with the same lysate to normalize the protein loading. Results shown are the representative of three independent experiments. The density of each band is measured and normalized to protein loading. The data obtained are analyzed and expressed as mean ± S.D. Expression of cyclin D3 (A), p21Cip1 and p15INK4b (B) are increased very significantly by I mM to 7 mM butyrate concentration vs. control (p < 0.001) and by 0.1 µM to 0.5 µM TSA concentration vs. DMSO (p < 0.001). Expression of cdk4 and cdk2 (A) are significantly decreased by 2 mM to 7 mM butyrate vs. control (p < 0.01) and by 0.25 µM to 0.5 µM TSA vs. DMSO (p < 0.01). Cdk6 is marginally affected by butyrate and TSA compared to their respective controls (A). Cyclin D3 (C), p21Cip1 and p15INK4b (D) are significantly increased both by 5 mM butyrate and 0.5 µM TSA vs. control (p < 0.001) at all experimental periods of time. Cdk4 and cdk2 are decreased by butyrate significantly after 6 h of treatment (p < 0.001) but no significant change in cdk6 vs. control (C). TSA causes significant reduction of cdk4 after 6 h (p < 0.001), cdk2 after 24 h (p < 0.05) and no significant effect on cdk6 (C) compared to control. Phosphorylation of Rb protein (D) is significantly inhibited both by butyrate and TSA after 6 h treatment compared to control (p < 0.001).