Fig. 4

The mutant haplotype reduced 13q-Enh enhancer activity by impairing the enhancer p53 response. a Diagram displaying the relative positions of the p53 binding sites and the three common SNPs in the 13q-Enh enhancer. b Western blotting confirmed the increased p53 protein level in Beas-2B transfected with p53 expression plasmids (left panel) and the decreased p53 protein level in Beas-2B cells with p53-knockdown (right panel). c Luciferase reporter assays showed significantly differential responsiveness of the wild-type allele and the mutant C-G-C allele in Beas-2B cells that either overexpressed p53 (left panel) or knockdown of p53(middle panel) which was stronger than that in the control. So did the responsiveness of wild-type and mutant enhancer in NNK-treated cells (right panel). The mutational effects were strengthened in response to p53 or in the case of NNK exposure. d Western blot indicated that NNK treatment significantly induced the expression of p53 in Beas-2B and MRC-5 cells. e Real-time PCR revealed that overexpression of p53 significantly increased the TNFRSF19 expression in Beas-2B wild-type cells, but had almost no effect on this gene expression in the 13q-Enh^−/− clones (left panel). Similarly, NNK also induced differential expression of the TNFRSF19 in wild-type Beas-2B cells and the 13q-Enh^−/− clones (right panel). f The 13q-Enh activity (left panel) and the TNFRSF19 expression (right panel) were also significantly enhanced in MRC-5 cells that overexpressed p53 or were treated by NNK