a) Real-time qPCR validation of fold change in ERAP1 mRNA expression in HCT116 ( p53 −/−) cells transfected with wild-type p53 (p53WT) or one of 6 p53 mutants, compared with cells transfected with the pcDNA3.1 control. ( b) Real-time qPCR analysis of relative basal ERAP1 mRNA expression levels in p53 +/+ and p53 −/− HCT116 cells. ** P<0.01 (two-tailed Student’s t-test). ( c) Western blot of ERAP1, ERAP2, p21, p53 and β-actin (loading control) in HCT116 ( p53 +/+) and HCT116 ( p53 −/−) cells. ( d) Dual-luciferase assay results of p53WT or the p53 dominant-negative mutant R175H co-transfected with different ERAP1 RE constructs (pGL3-pro-ERAP1-RE1 and RE2, sequences listed in the table at the bottom panel) into HCT116 ( p53 −/−) cells. Data are presented as percentage luciferase activity relative to the pcDNA3.1 control vector-transfected cells. Luciferase assay using p21 promoter construct (pGL3-pro-p21) serves as the positive control. ( e) Binding affinity of wild-type p53 (p53WT) or the six p53 mutants to the identified ERAP1 p53RE sequences (wild-type or mutant) was determined by ProLabel Protein–DNA binding assay. ( f) Schematic representation of identified ERAP1 p53RE (RE2) in relation to the ChIP-seq peaks and genomic localization of ERAP1 gene. TSS: transcription start site. Data are representative of three independent experiments and error bars represent s.d. of technical replicates (mean±s.d.), n=3, in a, b, d, e.