FIGURE 2.

MS-275 induces expression of IGFBP-3 in HepG2 cells. A, cells were treated with MS-275 for 24 h (triangles) or 48 h (squares), and IGFBP-3 was measured in conditioned media by radioimmunoassay. Data are means ± S.D. of triplicate determinations from one representative experiment of three. B, cells were treated with MS-275 for 24 h, and IGFBP-3 mRNA was measured by qRT-PCR. IGFBP-3 mRNA was normalized to the reference gene HMBS, and results shown are relative to IGFBP-3 expression in the absence of MS-275. Values are means ± S.D. of triplicate determinations in three separate experiments. C, ChIP analysis of IGFBP3 promoter acetylation in untreated and MS-275-treated HepG2 cells. Immunoprecipitations used RNA pol II antibody (lanes 1 and 5), control rabbit IgG (lanes 2 and 6), or antibodies against acetylated histone H3 (lanes 3 and 7) or acetylated histone H4 (lanes 4 and 8). PCR amplification of IGFBP3 promoter regions −2 to +134 and +68 to +254 is indicated. ChIP assays were performed three times with similar results. D, mean ± S.D. of three independent assays was calculated by densitometric analysis of imaged blots, with changes after MS-275 treatment shown relative to untreated controls (Ctrl). *, p < 0.002 compared with control, determined by ANOVA.