Figure 5. Inhibition of PAD4 and/or HDACs impacts on histone modifications at p53 target gene promoters and cancer cell growth in a p53-dependent manner.
(a–b) ChIP experiments were performed to analyze the level of p53, PAD4, HDAC2, histone H3 K9/K14 acetylation (H3K9/14Ac), H3 citrullination (H3Cit), or H3 Arg 17 methylation (H3R17Me) in U2OS cells before and after treatment with the PAD4 inhibitor Cl-amidine (200 μM) and/or the HDAC inhibitor SAHA (0.4 μM) at the p53 binding sites of p21 (a), GADD45 (b), and PUMA (b). ChIP signals were calculated as percentages of the input (% IP). Averages and standard deviations were shown (n=6). Cl-amidine was prepared via a solution-phase strategy following reported procedures (Causey and Thompson, 2008). The final compound was purified by silica gel flash chromatography. The structure and purity (>95%) was characterized by 1H-NMR and mass spectrometry. (c) The effect of PAD4 and/or HDAC2 inhibition on the expression of p21 in U2OS cells and U2OS/p53-shRNA cells was analyzed by western blot assays. β-actin was probed as a control to ensure equal loading. (d) The growth inhibitory effect of the PAD4 inhibitor Cl-amidine (200 μM), the HDAC inhibitor SAHA (0.4 μM), or both inhibitors was analyzed in U2OS cells. (e) The growth inhibitory effect of the PAD4 inhibitor Cl-amidine (200 μM), the HDAC inhibitor SAHA (0.4 μM), or both inhibitors was analyzed in U2OS/p53-shRNA cells. To obtain cell growth curve, 60,000 U2OS cells or U2OS/p53-shRNA cells were seeded in each well of the 6-well plate. Cl-amidine and SAHA were used at 200 μM and 0.4 μM, respectively, to treat cells singularly or in combination. The cell numbers were counted at the time points indicated after inhibitor treatment using a hemocytometer at time points indicated. The fold of cell number increase and standard deviations at each time point were obtained from three independent experiments.