Figure 6.

KLF4 regulation of FoxM1 expression in human gastric tumors. (A) Tissue sections were prepared from 86 formalin-fixed, paraffin-embedded human gastric tumor specimens. Immunostaining of the sections was performed using specific anti-KLF4 and -FoxM1 antibodies. FoxM1 expression levels were inversely correlated with KLF4 expression levels (P<001; χ² test). (B) Representative photographs of two cases showing KLF4 underexpression and FoxM1 overexpression. (C) Deletion mutants of FoxM1 promoter reporters were transfected into SK-GT5 cells in triplicate, and the relative promoter activities were measured 24 hours after transfection. (D) Schematic diagram of the FoxM1 proximal promoter. The nucleotide positions and sequences of the putative KLF4 binding site and PCR forward and reverse primers for ChIP analysis are shown (D1). Chromatin was extracted from SK-GT5 cells, and the ChIP assay was performed using a specific anti-KLF4 antibody and oligonucleotides flanking the FoxM1 promoter region containing the KLF4-binding site (D2). (E) N87 and SK-GT5 cells were transduced with a control Ad-EGFP (Neo) or Ad-KLF4 at an MOI of 10 or 20 for 24 hours. Total protein lysates were harvested from the cell cultures, and the levels of KLF4 (exogenous, as determined using an anti-FLAG antibody) and FoxM1 expression were determined using Western blot analysis (E1). SK-GT5 cells were transduced with Ad-KLF4 or Ad-EGFP at an MOI of 10 for 24 hours. Chromatin fragments were prepared for ChIP analysis using control IgG and an anti-Flag antibody (E2). The pFXM1-360 proximal promoter was transfected into SK-GT5 cells in triplicate with pcDNA3.1, a KLF4 expression vector, nontargeting control siRNA, or FoxM1-siRNA. The relative promoter activities were assessed 24 hours after transfection (E3).