Figure 5.

DNA-PK regulates EZH2 HMT activity in human CD8⁺ T cells. (a) Purified CD8⁺T cells isolated from PBMCs were stimulated with anti-CD3/CD28 antibodies plus IL-2. At different time points, cells subjected to DMSO or 5 μM DMNB treatment were immunoprecipitated with anti-EZH2 antibody, and this was followed by immunoblotting. (b) CD8⁺T cells were stimulated with anti-CD3/CD28 antibodies in the presence of different doses of DMNB and NU7441. Seventy-two hours later, cells were collected for immunoblotting. (c) CD8 ⁺T cells were stimulated with anti-CD3/CD28 antibodies in the presence or absence of GSK126 (2 μM) and DMNB (5 μM). Seventy-two hours later, cells were collected for immunoblotting. (d) CD8⁺T cells treated with 5 μM DMNB were collected for ChIP assays against H3K27me3 or IgG control. Total input DNA before immunoprecipitation was used for normalization of data. The graph shows the relative amount of H3K27me3 and IgG at the regions of Eomes, Bim, Dab2ip, MYT-1, and Gapdh. The results are the mean±S.E.M. from three independent experiments. *P<0.05. (e) Total RNA was isolated from CD8 ⁺T cells treated with 5 μM DMNB on day 3 post stimulation, and gene expression was determined by real-time PCR analysis. The results are the mean±S.E.M. from three independent experiments. *P<0.05