The increase in DNMT3a by TGF-β1 was inhibited by the protein synthesis inhibitor cycloheximide (CHX).
A, CCL210 cells were treated with TGF-β1 (2 ng/ml) for 4, 8, 24, and 48 h, and DNMT3a mRNA levels were assayed by real time RT-PCR (n = 4). B, CCL210 cells were treated with CHX (15 μm) for the indicated times, and DNMT3a protein was assayed by immunoblot. A representative immunoblot is shown, with the mean densitometric values of five independent experiments shown below. *, p < 0.05 relative to the 2-h time point with no CHX. C, cells were treated for varying times with CHX (15 μm) ± TGF-β1 (2 ng/ml), and expression of DNMT3a was compared relative to untreated cells at the 2-h time point (n = 3). *, p < 0.05. D, cells were treated for 8 or 16 h in the presence or absence of CHX (15 μm) and TGF-β1 (2 ng/ml), and expression of DNMT3a was assayed by immunoblot. The mean densitometric values relative to untreated control of seven independent experiments are shown below a representative immunoblot. *, p < 0.05. CHX, cycloheximide.