Fig. 4: p53 transcriptionally represses USP22 expression under H₂O₂ treatment.

A RKO and RKO E6 cells were treated H₂O₂ for the indicated time intervals, and cell lysates were subjected into immunoblotting using the indicated antibodies. B RKO and RKO E6 cells were treated H₂O₂ for 24 h, and USP22 mRNA expression was examined by qRT-PCR. GAPDH was used as an internal control. Values were normalized to control (mean ± s.e.m., n = 3 independent experiments, two-tailed Student’s t-test). ns, not significant. ^**P < 0.01. (C, D) RKO and HCT116 cells were transfected with siRNAs against p53, and USP22 protein and mRNA levels were analyzed by immunoblotting (C) and qRT-PCR (D), respectively. In D, values were normalized to control (mean ± s.e.m., n = 3 independent experiments, two-tailed Student’s t-test). ^**P < 0.05. E RKO cells were transfected with p53 siRNA, and then treated with H₂O₂ for 24 h. Cell lysates were subjected into immunoblotting using the indicated antibodies. F Chromatin immunoprecipitation (ChIP) assays demonstrate the binding of p53 to USP22 promoter in RKO cells under H₂O₂ treatment. Two potential p53 binding elements were predicted by hTFtarget database and indicated. The resultant DNA from ChIP was analyzed by PCR and then separated by agarose gel electrophoresis. IgG was used as an isotype control, and an irrelevant DNA sequence was used as a negative control.