Fig. 5. Effects of TSA treatment on the ectopic alphoid YAC sites. (A) 7C5HT1-19 cells were analyzed after 2 days of TSA treatment or after a further 7 days of culture without TSA, by western blotting using anti-acetylated histone H3 (upper) and H4 (lower) antibodies. (B) 7C5HT1-19 cells were treated with TSA and applied to TAU gel (left and middle) and SDS gel (right) and analyzed by western blotting with anti-acetylated histone H4 (Lys 12) or anti-CENP-A antibody. (C) Proportions of cells containing a minichromosome. Cells were treated with the indicated concentrations of TSA for 2 days and then cultured for 7 days without TSA. More than 50 metaphase cells were analyzed for each sample. Bars indicate s.e.m. (D) ChIP analysis of TSA-treated 7C5HT1-19 cells using anti-acetylated histone H3 antibody. HT1-19 cells were treated with 1 µg/ml of TSA for 2 days and then cultured for 7 days without TSA. Immunoprecipitated DNA was quantitated by real-time PCR using primers indicated in Fig. 4A. The bar chart shows the percentage of recovery by immunoprecipitation (% IP) using normal IgG or anti-acetylated histone H3 antibody. The colors of the columns denote the different DNA sequences as shown in lower panel. Bars indicate s.e.m. (E) Relative transcription levels of the bsr gene in TSA-treated cells were analyzed by real-time RT-PCR. The transcription levels of the ß-actin gene were not significantly changed by the TSA treatment in this analysis, so each level of bsr transcript was normalized to that of the ß-actin transcript. Bars indicate s.e.m.