Fig. 4

STIL associates with FOXM1 to enhance FOXM1-mediated transcriptional activity. a FOXM1 protein levels were analyzed by Western blotting in FOXM1-knockdown CL1-5 cells. Tubulin served as loading control. b–e SLUG mRNA levels were measured by qPCR method (b and d) and the SLUG promoter-driven luciferase activity was determined by reporter assay (c and e) in FOXM1-knockdown CL1-5 cells transiently transfected with indicated constructs (b and c) and CL1-0 cells transiently transfected with the indicated constructs (d and e). f The endogenous association between STIL and FOXM1 in CL1-5 cells was analyzed by co-IP analysis and Western blotting using the indicated antibodies. g The potential FOXM1 DNA-binding sites within the SLUG promoter (upper panel), and the binding of STIL and FOXM1 to the SLUG promoter were analyzed by ChIP-qPCR assay (lower panel) in CL1-5 cells. ARG2 promoter was served as the positive control for FOXM1-binding, and the region of 6.0 kb upstream of SLUG transcriptional start site within SLUG promoter was used as the unrelated control. h The binding of STIL to the SLUG promoter was examined by ChIP-qPCR assay in FOXM1-knockdown CL1-5 cells. i The mRNA levels of NANOG, SOX2, and POU5F1 were measured by qPCR method in FOXM1-knockdown CL1-5 cells transiently transfected with the indicated constructs. Data information: In g and h, Methylation of histone H3 (H3-K9Me3) on SAT2 gene was used as a positive control and IgG as a negative control for the ChIP-qPCR assay. Statistical data in b–e and g–i represent the mean ± SD (n = 3 independent experiments). Significance is determined by t-test (NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001)