Figure 7.

MS-275 withdrawal downregulates Snail expression, while maintaining an epithelial-like phenotype. (A) Effect of MS-275 withdrawal on MCs morphology and on Snail, αSMA and ZO-1 expression and localization. MCs were treated with MS-275 (250 nM) for 72 h. Then, the pharmacological inhibitor was washed out and the cells were cultured for three more days. MCs treated with MS-275 for three days were used as control. Cells were then processed for immunofluorescence using phalloidin, a monoclonal antibody against Snail, a monoclonal antibody against α-SMA and a polyclonal antibody against ZO-1. Cell nuclei are shown in blue (Hoechst 33342). Confocal images are shown from one representative experiment of three performed. (B) Effect of MS-275 withdrawal on epithelial and mesenchymal markers. Quantitative RT-PCR was performed on total RNA from MCs treated as above. Expression of Snail, MMP2, Col1A1, PAI-1, TGFβ1, TGFβRI, E-cadherin, Occludin was evaluated on total RNA by qRT-PCR. Data are reported as ratios of MS-275-treated and MS-275/withdrawal samples with respect to the non-treated samples. Bars represent means ± SEM of 5 experiments. (C) Western blots showing the expression of E-cadherin, Occludin and Snail in total cell lysates of MCs treated as above. GAPDH expression was used as a loading control. Representative experiment of 5 performed. (D) Proposed model describing the effect of MS-275 on Snail activity. Snail functional repressive activity (inhibition of E-cadherin expression) is HDAC1-dependent and thus is hampered by treatment with MS-275. P < 0.05 was considered significant.