Figure 6.

Treatment with MS-275 increases Snail expression while impairing its activity. (A) Effect of treatment with HDAC inhibitors over Snail expression in MCs. MCs were treated with DMSO vehicle (NT), with MS-275 (250 nM) or MC-2500 (250 nM) for 72 h. Samples were left untreated or were treated with TGFβ1 (2 ng/ml) for the last 24 h. Expression of Snail was evaluated on total RNA by qRT-PCR. n = 4. (B) Representative Western blot experiment of 5 performed showing expression of E-cadherin and Snail from cell lysates of MCs treated as above. GAPDH was used as a loading control. (C) Effect of MS-275 treatment on Snail expression and localization. MCs were treated with MS-275 (250 nM), or DMSO for 48 hr and then stimulated with TGFβ1 (2 ng/ml) for 24 h before processing for immunofluorescence with a monoclonal antibody against Snail. Cell nuclei are shown in blue (Hoechst 33342). Confocal images are shown from one representative experiment of three performed. (D) qPCR analysis of ChIP assays with anti-Snail (Snail) and anti-HDAC1 (HDAC1) antibodies and, as control, normal rabbit IgG (IgG) on chromatin from MeT5A cells treated with MS-275 for 72 h or with DMSO, and treated with TGFβ1 for 24 h (TGFβ1). Data show the enrichment of Snail and HDAC1 on Snail consensus binding site of human E-cadherin promoter. Values derived from 3 independent experiments are reported as means ± SEM and expressed as ((IP/IgG)/Input). Statistically significant differences are reported. (E) qPCR analysis of ChIP assays with anti-acH3 antibody and, as controls, normal rabbit IgG on chromatin from MeT5A cells treated with MS-275 for 72 h or left untreated (NT) and treated with TGFβ1 for 24 h (TGFβ1) when indicated. Data show the enrichment of H3 acetylation on Snail consensus binding sites of human E-cadherin promoter. Values derived from five independent experiments are reported as means ± SEM. and expressed as ((IP-IgG) %Input). Statistically significant differences are reported. P < 0.05 was considered significant.