FIGURE 1.

Direct physical binding of baicalein to TLR4 protein (A) Three‐dimensional ribbon model of the baicalein‐TLR4 complex. (B) Molecular dynamic and root‐mean‐square deviation values of the protein backbone showing the binding between baicalein and TLR4 during molecular stimulation. (C) Surface plasmon resonance showing the real‐time measurements of the binding affinity, (D) kinetics analysis and (E) the computer fitting of the binding of baicalein to TLR4 protein. (F) Bio‐layer interferometry analysis showing the binding between baicalein and TLR4. (G) Cellular thermal shift assay (CESTA) analysis of the binding between baicalein and TLR4 protein in CRC cells. (H) FITC‐conjugated LPS signals in TLR4‐overexpressed CRC cells. (I) Western blot showing the levels of p‐NF‐κB and p‐AKT (ser473) in the HCT116 and DLD‐1 cells. Cell viability of (J) HCT116 and (K) DLD‐1 in the presence of LPS (10μg/ml) after baicalein treatments. (L to Q) Viability of HCT116 cells, TLR4‐overexpressed HCT116 cells (TLR4‐116), DLD‐1 cells, and TLR4‐overexpressed DLD1 cells (TLR4‐DLD) after baicalein treatments. Shown is mean ± SE, n = 3 individual experiments, *P < 0.05, **P < 0.01 compared with control. a < 0.05, aa < 0.01 compared to LPS only. b < 0.05, bb < 0.01 compared with 24h and c < 0.05 compared with 48h at the indicated concentration. d < 0.05, dd < 0.01 compared to HCT116 or DLD‐1 cells under the same baicalein concentration. TLR4, Toll‐like receptor 4; LPS, lipopolysaccharides