Fig 2: FGF7 promotes the expression of E11 in primary osteoblasts and MC3T3-E1 cell line. (A) RNA sequencing results showing the changes of E11 in response to FGF7 in 24 h. The data were presented as the ratio of FPKM to the inner beta-actin levels. FPKM: Fragments per Kilobase of transcript per Million fragments mapped. Cell samples from each group were obtained from three independent cell isolates (n = 3). (B) Western blotting showing the protein expression of E11 in primary osteoblasts and MC3T3-E1 cell line after treatment of FGF7 (10 ng/ml) for 24 h. beta-actin was used as the loading control. (C) Quantification of western blot analysis in (B). The results were based on three independent experiments (n = 3). * p < 0.05, ** p < 0.01. (D) Representative IF staining by CLSM showing the increased expression of E11 in primary osteoblasts and MC3T3-E1 cell line in response to FGF7 (10 ng/ml). F-actin (green) and Dapi (blue) were used to counterstain the background. The white boxed areas are showing the distribution of E11 in single cells. The yellow boxed areas are showing the distribution of E11 at cell junctions. The white arrows are revealing the details of the distribution of E11. (E) Quantification of E11 fluorescent intensity in (D) by Image J. The results were based on three independent experiments (n = 3). * p < 0.05. (F) Quantification further showing the changes in cell junction numbers induced by FGF7. The results were based on three independent experiments (n = 3). * p < 0.05. (G) Bright field showing the cell morphology at the edge of the scratch wound after FGF7 induction in a concentration-dependent manner for 36 h in MC3T3-E1 cell line and the white arrows showing the elongated osteoblasts cell processes.

Fig 3: E11 is involved in the FGF7-induced functional GJIC and up-regulation of Cx43. (A) The scrape loading/dye transfer (SL/DT) assay showing functional GJIC in living primary osteoblasts treated with FGF7 (10 ng/ml) in the presence or absence of E11 siRNA. The Lucifer yellow dye enters cells at the scratch (dotted white lines) and is transferred to cells distant from the scratch (green arrows). Intercellular gap junction transfer was calculated by measuring the distance from the scratching edge to the most distant cells with Lucifer yellow uptake. The boxed area further showing the different transmission speeds. (B) Quantification showing different transmission speeds of Lucifer yellow in (A). The results were based on three independent experiments (n = 3). *** p < 0.001, **** p < 0.0001. (C) Western blotting showing the protein expression of Cx43 in primary osteoblasts treated with FGF7 (10 ng/ml) in the presence or absence of E11 siRNA. (D) Quantification of western blot analysis in (C). The results were based on three independent experiments (n = 3). ***p < 0.001, **** p < 0.0001.

Fig 4: FGF7 increases the expression of connexin 43 in primary osteoblasts and MC3T3-E1 cell line. (A) Western blotting showing the protein expression of Cx43 in primary osteoblasts and MC3T3-E1 cell line after treatment of FGF7 (10 ng/ml) for 24 h. beta-actin was used as the loading control. (B) Quantification of western blot analysis in (A). The results were based on three independent experiments (n = 3). ** p < 0.01. (C) Representative IF staining by CLSM showing the increased expression of Cx43 in primary osteoblasts and MC3T3-E1 cell line in response to FGF7 (10 ng/ml). F-actin (green) and Dapi (blue) were used to counterstain the background. The white boxed areas showing the distribution of Cx43 in single cells. The yellow boxed areas showing the distribution of Cx43 in cell junctions. The white arrows and dotted lines showing the details of the distribution of Cx43. (D) Quantification of Cx43 fluorescent intensity in (C) by Image J. The results were based on three independent experiments (n = 3). * p < 0.05. (E) Quantification further showing the changes in cell junction numbers induced by FGF7. The results were based on three independent experiments (n = 3). * p < 0.05.

Fig 5: The high expression of Fgf7 of osteoblasts and the distribution of FGF7 in bone tissue. (A) RNA sequencing results showing the whole fibroblast growth factor family members and receptor members of primary osteoblasts. The data were presented as log (FPKM + 1) and showed the higher mRNA expression of Fgf7 among other FGF ligand members. FPKM: Fragments per Kilobase of transcript per Million fragments mapped. The results were based on three independent samples (n = 3). (B) IHC showing the distribution of FGF7 in the femur of 4 weeks wild-type C57 mice. Fast green is used to counterstain the background. Boxed areas further showing that FGF7 is partly generated by osteoblasts on the surface of trabecular bone within the secondary ossification center (boxed area 1) and subchondral bone (boxed area 2).