Figure 5.

Overexpression of FoxM1 protects HCT-15 cells from thiostrepton-mediated inhibition, apoptosis, and inhibition of migration and invasion. A: Vector and FoxM1-transfected HCT-15 clones 1 and 3 were treated with 5 and 10 μmol/L thiostrepton for 48 hours. Cell proliferation assays were performed using MTT as described in Materials and Methods. Data are reported as means ± SD of three independent experiments, with replicates of six wells for all of the doses and vehicle control for each experiment. *P < 0.05, Student's t-test. B: Vector and FoxM1-transfected HCT-15 cells were treated with 5 and 10 μmol/L thiostrepton for 48 hours. Thereafter, the cells were washed, fixed, and stained with propidium iodide, and analyzed for DNA content by flow cytometry. At least three independent experiments were performed for all of the cell lines. *P < 0.05, Student's t-test. C: After treatment with 5 and 10 μmol/L thiostrepton for 48 hours, cells were lysed and proteins were separated on SDS-polyacrylamide gel electrophoresis and were immunoblotted with caspase-3, cleaved caspase-3, PARP, and β-actin. D: HCT-15 vector and HCT-15 clone 3 cells were transfected with scrambled siRNA and FoxM1 siRNA (100 nmol/L). After 48 hours, cells were lysed and proteins were immunoblotted with antibodies against caspase-3, cleaved caspase-3, PARP, and β-actin. HCT-15 vector and FoxM1-transfected HCT-15 clone 3 were treated with 5 and 10 μmol/L thiostrepton for 48 hours and activity of VEGF (E) and of MMP-2 (F) was determined by enzyme-linked immunosorbent assay. *P < 0.05, Student's t-test. G: HCT-15 vector and FoxM1-transfected HCT-15 clone 3 cells were treated with 5 and 10 μmol/L thiostrepton and invasion and migration assays were performed. H: After treatment with 5 and 10 μmol/L thiostrepton for 48 hours, cells were lysed and proteins were separated on SDS-PAGE and immunoblotted with FoxM1, MMP-2, MMP-9, and β-actin.