Fig. 4
From: The development of an anti-cancer peptide M1-21 targeting transcription factor FOXM1
M1-21 interacted with multiple regions of FOXM1 protein and inhibited FOXM1-related transcriptional activities. A GST-tagged recombinant proteins fused with different FOXM1 regions (100 µg) (GST-FOXM11 − 138, GST-FOXM1221 − 353, GST-FOXM1330 − 520, GST-FOXM1500 − 680, or GST-FOXM1688 − 748) were incubated with Biotin-M1-21 (20 µM) respectively. Streptavidin magnetic beads were added to the mixtures to pull down biotin-M1-21/protein complexes. The recombinant proteins in the samples were separated by PAGE-gel and detected with Ponceau S staining solution (marked by *). 10% of recombinant proteins (10 µg) were used as input controls. B The binding affinity of M1-21 to GFP-FOXM11 − 138 (GFP-M1(1-138)), GFP-FOXM1232 − 332 (GFP-M1(232–332)), or GFP-FOXM1688 − 748 (GFP-M1(688–748)) was measured by MST. Mean binding affinity values (KD (µM)) were shown on the panel. Experiments were repeated three times with similar results. C Reporter plasmids containing the luciferase gene downstream of the 6×FOXM1 binding sequence (6×FOXM1 Binding-Luc, 0.4 µg) or the − 1.8 kb CDC25B promoter (CDC25B pro(-1.8 kb)-Luc, 0.4 µg) were co-transfected with pCMV-FOXM1 (0.6 µg) into HEK-293T cells, plus pRL-CMV plasmid (20 ng/well) as a loading control. After 24 h, the cells were treated with M1-21mut (20 µM) or M1-21 (5, 10, 20 µM) for another 24 h. Then cell lysates were prepared and used for the measurement of dual Luciferase activities. Number of repetitions = 3. D The Flag-FOXM1 inducible MDA-MB-231 cell line (231-Flag-FOXM1-Ind) was induced with doxycycline (200 ng/mL) for 24 h and then treated with M1-21mut (20 µM) or M1-21 (10, 20 µM) for 6 h. Cell lysates (500 µg) were extracted and incubated with anti-Flag magnetic beads to pull down Flag-FOXM1/proteins complexes. The levels of Flag-FOXM1 and PLK1 proteins in the samples were detected by Western blotting. 5% of cell lysates (25 µg) were used as input controls. E Flag-FOXM1, LIN9, and B-MYB protein levels in panel D samples were detected by Western blotting. F Reporter plasmids containing the luciferase gene downstream of the − 1.4 kb PLK1 promoter (PLK1 pro(-1.4 kb)-Luc, 0.4 µg) were co-transfected with pCMV-FOXM1 (0.6 µg) into HEK-293T cells, plus pRL-CMV plasmid (20 ng/well) as loading control. After 24 h, cells were treated with M1-21mut (20 µM) or M1-21 (5, 10, 20 µM) for another 24 h. Then cell lysates were prepared and used to measure dual Luciferase activity. Number of repetitions = 3. G Flag-FOXM1 and β-catenin levels in panel D samples were detected by Western blotting. H MDA-MB-231 cells were treated with M1-21mut (20 µM) or M1-21 (20 µM) for 6 h. Cytosol and nuclear protein samples were prepared and β-catenin levels were detected by Western blotting. β-tubulin or Lamin B1 was used as the loading controls for cytosol or nuclear proteins respectively. I TCF/LEF Binging-Luc reporter plasmid (0.4 µg) was co-transfected with pCMV-FOXM1 (0.6 µg) into HEK-293T cells, plus pRL-CMV plasmid (20 ng/well) as loading control. After 24 h, cells were treated with M1-21mut (20 µM) or M1-21 (5, 10, 20 µM) for another 24 h. Then cell lysates were prepared and used to measure dual Luciferase activity. Number of repetitions = 3. J MDA-MB-231 cells were treated with M1-21mut (20 µM) or M1-21 (20 µM) for 24 h. The mRNA levels of PLK1, CDC25B, and Vimentin (VIM) were measured by qPCR. GAPDH was used as a loading control. K MDA-MB-231 cells were treated with M1-21mut (20 µM) or M1-21 (20 µM) at different time points (1.5, 3, 6, 9, 12, 24, 36 h). The protein levels of PLK1, CDC25B, and Vimentin (VIM) were measured by Western blotting. β-actin was used as a loading control. L The diagram depicts the molecular mechanisms of M1-21 inhibiting FOXM1-related transcriptional activities. mRNA values represented the mean ± SD of three replicates, and significance was calculated using unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001