Fig. 5: M1-20 inhibited FOXM1-related transcriptional activities.

A MDA-MB-231 cells were seeded in 6 cm plates for 12 h and then treated with M1-20 (10 µM) containing 10% biotin-labeled M1-20. After treatment for 24 h, the cells were harvested for the separation of the cytoplasmic and nuclear proteins. The levels of M1-20 in the cytoplasm (C) or the nucleus (N) were analyzed by Western blotting. α-Tubulin or Lamin B1 was used as a cytoplasmic or nuclear marker, respectively. B The EMSA experiment was performed by recombinant His-FOXM1 proteins (2 µM) and the FAM-labeled DNA probe (50 nM). M1-20 was added to the reactions with increasing concentrations (5 µM and 10 µM). The Cold probe (100×, 5 mM) was unlabeled with FAM to show the specificity of FOXM1/DNA complex formation. The reactions were performed in 4% native polyacrylamide gel electrophoresis in 0.5×TBE buffer and imaged with Kodak 4000 MM Imaging System (EX: 465 nm, EM: 535 nm for FAM). C Hela cells were transfected with pFlag-FOXM1 (6 µg) for 48 h. Cell lysates were harvested and respectively incubated with Flag magnetic beads, added to different dosages of biotin-labeled M1-20 (0 μg, 2 μg, 4 μg, 8 μg). Flag-FOXM1/protein complexes were analyzed by Western Blotting with certain antibodies. 10% of cell lysates (50 μg) were used as input controls. The levels of protein were quantified by Image J software. D The −1.4 kb PLK1 promoter-luciferase reporter plasmid (1 µg) and pRL-CMV plasmid (20 ng/well) were co-transfected with pFOXM1 (0.3 µg) into Hela cells. 12 h later M1-20 (20 µM), and M1-20mut (20 µM) were respectively added to the selected transfections. Then cell lysates were prepared 24 h later and used for the measurement of dual Luciferase activities. n = 3 for each group, ****P < 0.0001, two-tailed unpaired Student’s t-test. E HEK293T cells were transfected with pFlag-FOXM1 (6 µg) and 48 h later the cells were collected. The cell lysates (500 μg) were incubated with Flag magnetic beads and added with different quality of M1-20 (0 μg, 2 μg, 4 μg, 8 μg) to immunoprecipitation Flag-FOXM1/protein complexes. CBP, Biotin, and Flag proteins in samples were detected by Western Blotting with certain antibodies. 10% of cell lysates (50 μg) were used as input controls. F Reporter plasmids containing the 6×FOXM1 binding sequence (1 µg) or the −1.8 kb CDC25B promoter-luciferase reporter (1 µg) were co-transfected with pFOXM1 (0.3 µg) into HEK293T cells, plus pRL-CMV plasmid (20 ng/well) as a loading control. 12 h later M1-20 (20 µM) or M1-20mut (20 µM) was added to the selected transfections. Another incubation for 24 h later cell lysates were prepared for the measurement of dual Luciferase activities. n = 3 for each group, **P < 0.01, two-tailed unpaired Student’s t-test. G MDA-MB-231 cells were treated with M1-20 (10 µM) or M1-20mut (10 µM) and 24 h later the cells were collected for the preparation of total RNA. The mRNA levels of CDC25B and PLK1 were examined by RT-RCR. n = 3 for each group, ****P ≤ 0.0001, two-tailed unpaired Student’s t-test. H The diagram depicting the molecular mechanisms of M1-20 inhibiting the FOXM1-related transcriptional activities.