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. 2018 Jan 25;8:1595. doi: 10.1038/s41598-018-20043-7

Figure 5.

Figure 5

Butyrate promotes p53-binding to the CDH1 locus in EMT-resistant cells without affecting their integrity. (A) A549 cells, MCF7 cells, HMLE prep#2 cells, and BJ cells were subjected to ChIP using antibodies against the indicated modified histones, followed by quantitative RT-PCR analysis with the primers located at the ′b′ region of CDH1. Data are means ± SD of 3 independent experiments. *P < 0.05 (Student t-test). (B) MCF7 cells or BJ cells were treated with the indicated concentrations of butyrate for 24 h. 1.5 µg of chromatin fractions of these cells or non-treated A549 cells were subjected to immunoblot analysis with the indicated antibodies. An uncropped image of this immunoblot of A549 chromatin is also used in Supplementary Figure S4B. (C) ChIP was performed using non-treated (NT) or butyrate-treated (10 mM, 48 h) MCF7 cells or BJ cells with a normal rabbit IgG or anti-H3K27ac antibody, followed by quantitative RT-PCR analysis with primers located at the ′b′ region of CDH1. Data are means ± SD from 3 independent experiments. **P < 0.01, *P < 0.05; NS, not significant (Student t-test). (D) ChIP was performed using non-treated (NT) or butyrate-treated (10 mM, 48 h) MCF7 cells or BJ cells with a normal rabbit IgG or anti-p53 antibody, followed by quantitative RT-PCR analysis with primers located at the ′b′ region of CDH1. Data are means ± SD from 3 independent experiments. *P < 0.05, NS, not significant (Student t-test). (E) MCF7 cells transfected with scramble (Scr) or p53 (#1 or #2) siRNA were then treated with or without (NT) 10 mM butyrate for 24 h. Whole cell extracts were then subjected either to immunoblot analysis with antibodies to E-cadherin and β-actin, or to immunoprecipitation with antibodies to p53 (mouse monoclonal) before being subjected to immunoblot analysis with antibodies to p53 (rabbit monoclonal). Quantitative western blotting of E-cadherin and β-actin (E-cad and actin, respectively) was performed with the infrared fluorescence imaging system on an Odyssey imager, and normalized E-cad/actin ratios are indicated. Western blots were cropped for clarity; uncropped images are shown in Supplementary Figure S5.