Fig. 2

M1-21 was more stable than TAT-106-126 and inhibited multiple types of cancer cells. A TAT-106-126 peptide (30 µg) or D-retro-inverso M1-21 peptide (30 µg) was incubated with trypsin (0.1 µg), collagenase (0.1 µg), proteinase K (0.5 µg), mouse serum (20 µg) or cell lysates (20 µg) for 2 h. SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining were performed to measure peptide stability. B MDA-MB-231 cells were treated with biotin-labeled TAT-106-126 (20 µM) or biotin-labeled M1-21 (20 µM) for 2 h and then replaced with fresh medium. Cells were collected at different post-treatment time points (0, 1, 2, 4, 8, 16, 24, 36, 48 h). The protein lysates were extracted and detected by Western Blotting. β-actin was used as a loading control. C The mean IC50 value (µM) of TAT-106-126 or M1-21 at 48 h to MDA-MB-231 cells was obtained according to the procedure described in Fig. 1C. D The docking of peptide P22 (106–126) to the FOXM1 C-terminus (PDB ID 6OSW) was created by Rosetta FlexPepDock and PyMOL. Left, the interface of peptide-protein interaction, the peptide was shown in cyan, and the C-terminus of FOXM1 was shown according to its electrostatic potential. Right, hydrogen bonds are formed by residues at the interface of peptide and protein interaction. E Biotin-labeled M1-21 (Biotin-M1-21, 20 µM) or biotin-labeled M1-21mut (Biotin-M1-21mut, 20 µM) was added to MDA-MB-231 cell lysates (500 µg). Streptavidin magnetic beads were added to lysates to pull down the biotin-peptides/protein complexes. Biotin and protein in the samples were detected by Western blotting. 5% of cell lysates (25 µg) were used as input controls. F MDA-MB-231 cells were treated with Biotin-M1-21 (20 µM) for 4 h. Cytosol and nuclear protein samples were prepared and detected by Western blotting. β-tubulin or Lamin B1 was used as the loading controls for cytosol or nuclear proteins respectively. G The mean IC50 value (µM) of M1-21 at 72 h for multiple cancer cell lines was obtained according to the procedure described in Fig. 1C. M1-21mut was used as the control. Cell lines tested included breast cancer MDA-MB-231 and ZR-75-30, lung adenocarcinoma A549, colon cancer HCT116, renal clear cell tumor 786-O, cervical cancer Hela, bladder cancer 5637, glioma U251