Fig. 2
(-)-Epicatechin acts downstream of FGFR3 on cartilage homeostasis. a, b, c Representative western blots of tyrosine-phosphorylated FGFR3 (p-Tyr) expression from FGFR3 immunoprecipitated from HEK293 cells transfected with FGFR3Y373C (a), FGFR3G380R (b), and FGFR3K650E (c) exposed or not to (-)-epicatechin. d–g Computational results of (-)-epicatechin binding to FGFR3. In all these images, the protein is displayed as ribbons (yellow: β-sheet, red: α-helix, pink: turn and white: random coil), and the ATP active site is presented as a molecular surface in ice blue. (-)-Epicatechin is displayed in CPK colors with carbon in green. Structures on the top (d, top) correspond to the FGFR3-(-)-epicatechin complex after molecular docking. The hydrogen bonds between interacting residues (presented with gray carbon atoms) are shown with dashed lines. The structure on the bottom (d, bottom) was obtained after molecular dynamics and hyperdynamics simulations. e Detailed illustrations of the dissociation of (-)-epicatechin from FGFR3 kinase. From left to right, snapshots were extracted after 0, 5, 10, and 50 ns of classical molecular dynamics and after 40 ns of hyperdynamics (see Supplemental Movie 1). f, g Representative structures of (-)-epicatechin complexed with the kinases ERK1 (f), ERK2 (Supplementary Fig. 4) and p38 (g). The complexes obtained after molecular docking (i.e., static states) are shown on the left, and those obtained after the molecular dynamics and hyperdynamics simulations (i.e., dynamic states) are shown on the right. The ATP-binding sites are presented as molecular surfaces in ice blue. h Representative western blots of the expression levels of p-ERK1/2 and ERK1/2 in primary chondrocytes isolated from the femoral cartilage of Fgfr3Y367C/+mice. i Changes in the ratio of p-ERK1/2 to ERK1/2 (n = 5). j Representative western blots of the expression levels of p-p38 and p38 in primary chondrocytes isolated from the femoral cartilage of Fgfr3Y367C/+mice. k Changes in the ratio of p-p38 to p38 (n = 5). Data are presented as the mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1 by two-tailed, unpaired t test. The gray density analysis for the western blot data was performed by using ImageJ software
