Fig. 3
From: FoxM1 transactivates PTTG1 and promotes colorectal cancer cell migration and invasion
FoxM1 binds to and activates PTTG1 promoter. a Luciferase reporter assay. SW620 cells were transfected with pGL3-Basic, pGL3-PTTG1 –1300, −900, −600 or −300/+50 reporter plasmids and co-transfected with pcDNA3.1 control, pcDNA3.1-FoxM1 plasmid (FoxM1), siRNA control or FoxM1 siRNA (siFoxM1), together with Renilla reporter vector. Cells were harvested after 24 h and luciferase activities were examined. After normalizing to Renilla, results are expressed as mean ± SD. Assays were performed in triplicate and experiments were repeated three times. Results of a representative experiment are shown. *p < 0.05, **p < 0.01, ***p < 0.001. b FoxM1 binding sites on the -600 bp PTTG1 promoter (pGL3-PTTG1-600) are mutated and these mutants are co-transfected with FoxM1 to evaluate effects of FoxM1 on the activity of mutated promoter (1st site: −391 to −385, 2nd site: −359 to −353 bp of TSS). c Biotin-streptavidin EMSA. Representative EMSA shows the bindings of FoxM1 protein with Biotin-Probe in lane 2 (biotin-labeled 60 bp probe, containing both −391 to −385 and −359 to −353 bp wild type FoxM1-binding PTTG1 promoter motifs), with Biotin-dmProbe in lane 4 (biotin-labeled 60 bp probe, containing both −391 to −385 and −359 to −353 bp mutant FoxM1-binding sites) and with 1st site mProbe (biotin-labeled 60 bp probe with −391 to −385 bp mutant FoxM1-binding site, lane 5). Competition assays were performed using a 100-folds excess of cold probe (unlabeled 60 bp wild type probe, lane 3), 1st site oligo (unlabeled 22 bp oligonucleotide only containing −391 to −385 bp wild type FoxM1-binding motif, lane 6) and 2nd site oligo (unlabeled 25 bp oligonucleotide only containing −359 to −353 bp wild type FoxM1-binding motif, lane 7). d ChIP assay showing specific endogenous FoxM1 binding to the human PTTG1 promoter. About 500 bp length chromatin DNA in HCT116 cells were immunoprecipitated with FoxM1 antibody, negative control IgG, positive control histone H3 antibody (H3) and water blank control as indicated. Enrichment of chromatins was obtained with primers targeting PTTG1 promoter but not with primers targeting PTTG1 exon 2. The experiment was repeated twice. As VEGF is a known target of FoxM1, primers targeting VEGF promoter were included as a positive control