Fig. 1
From: The development of an anti-cancer peptide M1-21 targeting transcription factor FOXM1
Screening of TAT-106-126 peptide targeting FOXM1 protein. A An in silico peptide library (P1 to P24, 21-mer) was created from the FOXM1 N-terminus sequence (1-138aa) with a 5 amino acid shift window. The peptide candidates binding to FOXM1 C-terminus (PDB ID 6OSW) were selected by Rosetta FlexPepDock simulation and InterfaceAnalyzer calculation according to docking free energy (dG). B P20, P21, and P22 fused with the cell-penetrating TAT sequence (TAT-96-116, TAT-101-121, and TAT-106-126) were produced by solid-phase peptide synthesis. The binding affinity between the synthesized peptides and GFP-FOXM1688-748 (GFP-M1(688–748)) was measured by Microscale Thermophoresis (MST) (Monolith NT.115, NanoTemper). Data points indicated the fraction of GFP-M1(688–748)-bound peptides (ΔFNormal/Amplitude) at different concentrations and curves indicated the calculated fits. The error bars represented the standard error of three independent measurements. Mean binding affinity values (KD (µM)) were shown on the panel. Experiments were repeated three times with similar results. C MDA-MB-231 cells (2 × 10^5 cells/well) were seeded in 24-well plates for 12 h and treated with different concentrations of TAT-96-116, TAT-101-121, or TAT-106-126 (0, 20, 40, 60, 100, 150 µM). 48 h Later, trypan blue (0.4%) was added to each well for 3 min and the cells were fixed to 4% paraformaldehyde for imaging. The number of viable cells was counted by ImageJ software to calculate the cell viability of each well. The correspondence between cell viability and peptide concentration was plotted by GraphPad software. The mean IC50 value (µM) for each peptide was obtained from three replicates. D The TAT-106-126 peptide was biotin-labeled and added to 293T cell lysates (500 µg) expressing exogenous Flag-GFP-FOXM1(688–748) protein. The lysates were incubated with Streptavidin magnetic beads to pull down biotin-peptide/protein complexes. Biotin and Flag-GFP-FOXM1(688–748) protein in samples were detected by Western Blotting. 5% of cell lysates (25 µg) were used as input controls. E Biotin-labeled TAT-106-126 or TAT was added to 293T cell lysates (500 µg). The lysates were incubated with Streptavidin magnetic beads to pull down biotin-peptide/protein complexes. Biotin and endogenous FOXM1 protein were detected by Western Blotting. 5% of cell lysates (25 µg) were used as input controls