Figure 5

P53 is necessary for NF-κB-induced miR-34a transcriptional activity but not for NF-κB binding. (a) KYSE450 cells were transfected with pCDNA3.1or p65 in combination with the wildtype miR-34a promoter (P1). pRL-TK was used as transfection control. Cells were lysised and luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (b) pCDNA3.1or p65 and siRNA control or sip53 along with the wildtype miR-34a promoter were cotransfected in EC109. pRL-TK vectors were used as transfection control. Luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (c) EMSA was performed with nuclear extracts from KYSE450 cells using probe corresponding to the third κB site (34a3ΚB) in miR-34a promoter region. Competition and supershift assays against anti-p53, p65 and p50 antibody were also shown. (d) Chromatin derived from KYSE450 cells transfected with p65 or control vectors were immunoprecipitated with anti-p65, p50, p53 and IgG antibodies. Relative enrichment of each transcription factor-bound DNA was detected by qPCR using 34aΚB3 primers. Shown were results normalizing to input DNA.