recombinant human fgf9  (R&D Systems)

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: Mechanical stretch stress promoted maxillary development and reduced FGF9 expression in osteocytes. a) Schematic diagram of experiments in this section. b) The 3D analysis images of normal maxilla and underdeveloped maxilla under masticatory muscle strength and its quantification. c) Sagittal section of CBCT measuring the maxillary length (ANS‐PNS) of patients before and after facemask protraction and its maxillary growth quantification (untreated n = 20, facemask treated n = 18). d) Top 20 genes that were specifically expressed in osteocytes. e) Dural immunofluorescence images of DMP1 and FGF9 in human and mouse maxilla and its colocalization quantification. f,g) Micro‐CT reconstructed images of mice after facemask protraction for 1 or 2 week and quantification of maxillary length and maxillary percentage. h,i) The mRNA level of Runx2 , Bmp4 , and Fgf9 in the maxilla of mice after 2‐week facemask protraction. j) The representative images of MASSON and TRAP staining of maxilla after facemask protraction. The red arrow represents TRAP staining regions. k,l) Representative immunofluorescence images and its quantification of FGF9 in the maxilla of mice after facemask protraction. Scale bar = 50 µm. Data are presented as mean ± SD. Statistical comparison between groups was performed using an independent samples t ‐test. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***. Facemask protraction n = 6.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: Expressing, Immunofluorescence, Micro-CT, Staining, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: Mechanical stress reduced FGF9 expression in osteocytes and promoted osteogenesis in preosteoblasts. a) Schematic diagram of experiments in this section. b–d) The mRNA and protein level of FGF9 in osteocytes were quantified by qPCR, Western blot, and ELISA after applying 20% cyclic stretch for 12 or 24 h. e–g) Osteogenic differentiation of MC3T3 following co‐culture with osteocytes’ mechanical stress conditional medium: e) qPCR analysis of Runx2, Bmp4, Oc , and Alp mRNA expression; f) Representative immunoblot images and quantification of RUNX2 protein levels; g) Representative ALP staining images. h) The mRNA level of Fgf9 in osteocytes after siRNA knockdown. i–k) Osteogenic differentiation of MC3T3 following co‐culture with osteocytes’ FGF9 knockdown conditional medium: i) qPCR analysis of Runx2, Bmp4, Oc , and Alp mRNA expression; j) Representative immunoblot images and quantification of RUNX2 protein levels; k) Representative ALP staining images. l) The mRNA level of Fgf9 in knockdown osteocytes after 20% cyclic stretch stress. m–o) Osteogenic differentiation of MC3T3 following co‐culture with osteocytes’ FGF9 knockdown and mechanical stress conditional medium: m) qPCR analysis of Runx2, Bmp4, Oc , and Alp mRNA expression; n) Representative immunoblot images and quantification of RUNX2 protein levels; o) Representative ALP staining images. Scale bar = 100 µm. Data are presented as mean ± SD. n = 3. Statistical comparison between groups was performed using paired t‐test and independent samples t‐test. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Staining, Knockdown, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: FGF9 inhibited osteogenesis and neonatal maxillary bone remodeling in vitro. a) Schematic diagram of experiments in this section. b–d) Osteogenic differentiation of MC3T3 after 0–50 ng /mL recombinant FGF9 protein stimulation: b) qPCR analysis of Runx2, Bmp4, Oc , and Alp mRNA expression; c) Representative immunoblot images and quantification of RUNX2 protein levels; d) Representative ALP staining images. e) Representative immunohistochemical images for RUNX2 and BMP4 of neonatal maxilla following treatment with 25 ng/mL recombinant FGF9 protein or 1 µl/mL FGF9 neutralizing antibody. The dashed boxes represent magnified images. f) Von Kossa and TRAP staining of neonatal maxilla following treatment with 25 ng/mL recombinant FGF9 or 1 µl/mL FGF9 neutralizing antibody. Blue arrows indicate the mineralized staining areas, while red arrows denote the TRAP staining regions. g) Quantification of RUNX2, BMP4, Von‐kossa and TRAP staining. Scale bar = 50 µm. Data are presented as mean ± SD. n = 3. Statistical comparison between groups was performed using an independent samples t ‐test. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: In Vitro, Recombinant, Expressing, Western Blot, Staining, Immunohistochemical staining, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: FGF9 negatively regulated osteogenesis through ATF5 and NR2F1 independent of PI3K‐Akt, HIF‐1, and mTOR signaling pathway. a,b,j) RNA sequencing from MC3T3 treated with 25 ng/mL FGF9 for 24 h: a) Volcano plot and clustering analysis. b) Heatmap of differential transcription factors. j) KEGG pathway analysis. c) mRNA expression of Nr2f1 and Atf5 in preosteoblasts cultured with conditioned medium from osteocytes subjected to 20% mechanical stress. d–i) Osteogenic differentiation of MC3T3 after siRNA‐mediated knockdown of Nr2f1 and Atf5 : d,f) mRNA levels of Nr2f1 and Atf5 . e,g) qPCR analysis of Runx2, Alp, Bmp4 and Col1a1 mRNA expression. h) mRNA levels of Fgfr2 . i) Representative ALP staining images and its quantification. k) mRNA expression of osteogenic markers Runx2, Col1a1 and Fgfr2 in MC3T3 following treatment with FGF9 and corresponding signaling pathway inhibitors of PI3K‐Akt, HIF ‐ 1 and mTOR. Scale bar = 100 µm. Statistical comparison between groups was performed using an independent samples t‐test. Data are presented as mean ± SD. n = 3. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: RNA Sequencing, Expressing, Cell Culture, Knockdown, Staining, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: FGF9 interacted with FGFR2, inducing its nuclear translocation and resulting in an overall decrease in FGFR2 level. a) AlphaFold2‐based structural prediction of the binding interface between mouse FGF9 and FGFR2. b) Dual immunofluorescence staining and quantification of MC3T3 treated with 25 ng/mL recombinant FGF9 protein for 5 min. Dashed boxes indicate magnified images. Scale bar = 25 µm. c–f) mRNA and protein expression of FGFR2 in MC3T3 following co‐culture with osteocytes or treatment with recombinant FGF9: c) co‐culturing with osteocytes’ mechanical stress conditional medium. d) co‐culturing with osteocytes’ FGF9 knockdown conditional medium. e) co‐culturing with osteocytes’ FGF9 knockdown and mechanical stress conditional medium. f) culturing with 0–50 ng/mL recombinant FGF9 protein. g) Representative immunohistochemical images of FGFR2 in neonatal mouse maxilla following treatment with 25 ng/mL FGF9 recombinant protein or its 1 µl/mL neutralizing antibody. Scale bar = 50 µm. h) Dual immunofluorescence staining of MC3T3 treated with 25 ng/mL recombinant FGF9 protein for 15 min. Scale bar = 25 µm. i) Representative immunofluorescence images of FGFR2 in MC3T3 after 24 h of 25 ng/mL FGF9 treatment. Dashed boxes indicate magnified images. Scale bar = 50 µm. j) Western blot analysis and quantification of cytoplasmic and nuclear FGFR2 in MC3T3 after 48 h of 25 ng/mL FGF9 treatment. k) Dual immunofluorescence staining of FGFR2 with FBL or LAMP1 in MC3T3 after 3 and 6 h of 25 ng/mL FGF9 treatment. White arrows indicate the regions of co‐localization. Scale bar = 25 µm. l) Western blot analysis of nucleolar FGFR2 in MC3T3 after 48 h of 25 ng/mL FGF9 treatment. m) mRNA expression of the 45s rDNA precursor in MC3T3 following 25 ng/mL FGF9 treatment for 24 h. Data are presented as mean ± SD. n = 3. Statistical comparison between groups was performed using an independent samples t ‐test. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: Translocation Assay, Structural Proteomics, Binding Assay, Immunofluorescence, Staining, Recombinant, Expressing, Co-Culture Assay, Knockdown, Immunohistochemical staining, Western Blot, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: Overexpression of FGF9 suppressed maxillary development through the inhibition of osteogenesis and activation of osteoclastogenesis. a) Schematic diagram of experiments in this section. b) The ANB angles in patients with normal maxilla versus those with maxillary underdevelopment. c) Representative immunohistochemical images and quantification of FGF9 and FGFR2 expression in normal maxilla and underdeveloped maxilla. d–h) Overexpressing FGF9 through bone‐targeted adenovirus ( n = 6): d) Representative 3D Micro‐CT reconstruction images of skulls, with magnified views of the maxillary region and the incisor area. e,f) The quantification of maxillary length, mandible width and length, skull length, and the incisors’ overjet. g) Quantification of BV/TV and BS/BV in the premaxilla and maxilla. h,j) Representative images and quantification of H&E, Masson, and TRAP staining of the maxilla. i,k) Representative immunofluorescence images and quantification of RUNX2, BMP4, OCN, and FGFR2 expression in the maxilla. Scale bar = 50 µm. Data are presented as mean ± SD. Statistical comparison between groups was performed using an independent samples t ‐test. Statistical significance was determined as follows: p < 0.05*, p < 0.01 **, p < 0.001***.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: Over Expression, Inhibition, Activation Assay, Immunohistochemical staining, Expressing, Micro-CT, Staining, Immunofluorescence, Comparison

Journal: Advanced Science

Article Title: FGF9–FGFR2 Signaling via Osteocytes‐Preosteoblasts Crosstalks to Mediate Mechanotransduction‐Driven Intramembranous Osteogenesis in the Underdeveloped Maxilla

doi: 10.1002/advs.202506954

Figure Lengend Snippet: Schematic diagram of the study. Abnormally high FGF9 expression inhibits osteogenic differentiation, leading to maxillary underdevelopment. Insufficient mechanical signaling in the underdeveloped maxilla further drives FGF9 overexpression. Conversely, supplementing mechanical signals reduces FGF9 secretion, thereby promoting osteogenic differentiation and facilitating maxillary development. FGF9, secreted by osteocytes, binds to FGFR2 receptors on preosteoblasts, triggering the nuclear translocation of FGFR2. Nuclear FGFR2 modulates the transcription factors ATF5 and NR2F1, which in turn downregulate FGFR2 protein expression and activate downstream signals that inhibit osteogenesis.

Article Snippet: Within the corresponding groups, either 25 ng/mL recombinant FGF9 protein (Cusabio, CSB‐AP004131MO) or 1 μg/mL FGF9 neutralizing antibody (Bioss, bs‐5906R) was added.

Techniques: Expressing, Over Expression, Translocation Assay