Fig. 4

C/EBPα binding the SPOP proximal promoter region is abrogated by DNA methylation and induces SPOP expression in lung cancer cells. a Location of the predicted C/EBPα binding sites in the SPOP gene promoter region. The sequences depicted in boldface and underlined denote the predicted binding sites of C/EBPα, and the TSS is labeled. b ChIP assay demonstrating the direct binding of C/EBPα to the SPOP gene promoter in NSCLC tissues. The ChIP-enriched DNA fragments of the SPOP gene promoter using IgG and an anti-C/EBPα antibody were amplified by PCR. Total input was used as a positive control. c Sequential deletion and substitution mutation analyses identified C/EBPα binding sites in the SPOP gene promoter region. Serially truncated and mutated SPOP gene promoter constructs were cotransfected with siC/EBPα or siControl into A427 cells, and the relative luciferase (luc.) activities were determined. Following the transfection of the C/EBPα expression plasmid (d) or siC/EBPα (e) into the A427 and H1299 cells, the C/EBPα and SPOP mRNA and protein levels were determined by qPCR and western blotting. The data are presented as the mean ± SD of three independent experiments. *P < 0.05