Figure 6. Regulation of p53K370 methylation by SETDB1.

(a) To confirm that endogenous SETDB1 methylates endogenous p53, HCCLM3 cells stably infected with inducible SETDB1 shRNA were treated with doxycycline for 3 days, and then exposed to Doxorubicin for 24 h. Methylation of p53 was assessed using the antibody against p53K370me2. (b) SNU182 cells stably infected with inducible SETDB1 shRNA were treated with or without 1 μg ml⁻¹ doxycycline for 3 days after transfection and then exposed to 0.05 μg ml⁻¹ Doxorubicin for 24 h. The cells were harvested for western blot analysis using p53K370me2 antibody (1.2 μg ml⁻¹) alone or competed with 5 μM p53K370 unmethylated or monomethylated peptides or di-methylated peptide at the dose of 1, 5 and 10 μM. All competing peptides were of 31-mer long in length. (c) p53 null HCT116 cells were transfected with wild-type p53 or a mutant p53 with K370 replaced with A (p53K370A). Cells were also treated with 0.05 μg ml⁻¹ Doxorubicin for 24 h and harvested for western blot analysis using p53K370me2 antibody. GAPDH was analysed as the control. (d) p53 null HCT116 cells were transfected with wild type or p53R249S mutant together with SETDB1 or the set domain-deleted SETDB1 control. The methylation of p53 at K370 was measured by western blot analysis. (e) In vitro p53 methylation assays were performed using the endogenous SETDB1 complex pulled down from HCCLM3 cells that were treated with 0.5 μg ml⁻¹ of Doxorubicin for 6 h. The immunoprecipitated complex was used as the enzyme in the assay with full-length p53 protein as the substrate and S-adenosyl methionine (SAM) as the methyl donor. Methylation was assayed using western blot analysis. The reaction was carried out at room temperature or at 37 °C for the duration as indicated.