Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 1 Effect of LGR4 on bone formation in BMP9-KO mice. (A) The micro-CT images showing BMP9’s effect on bone formation. (B) Quantitative results of micro-CT assay showing BMP9’s effect on bone formation. (C) The effect of BMP9 on LGR4 in BMSCs from 4-week-old WT and BMP9-KO mice cultured in an osteogenic medium for 7 days shown by Western blotting. (D) The effect of BMP9 on bone formation shown by H&E staining. (E) The effect of BMP9 on LGR4 in femurs from 4-week-old WT and BMP9-KO mice shown by immunohistochemistry staining. (F, G) The effect of LGR4 on the ALP activity of BMSCs shown by histochemical staining. (H) The effect of LGR4 on the mineralization of BMSCs shown by histochemical staining. BV/TV, the ratio of bone volume to total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. *P < 0.05, **P < 0.01; n Z 6.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Micro-CT, Cell Culture, Western Blot, Staining, Immunohistochemistry, Activity Assay

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 2 Effect of BMP9 on LGR4 in multiple progenitor cells. (A) The level of LGR4 in the progenitor cells shown by Western blotting. (B) The effect of osteogenic BMP9 on LGR4 shown by Western blotting. (C) The effect of BMP9 on LGR4 over time shown by Western blotting. (D) The images of C3H10T1/2 cells showing the transfection rates of BMP9 at 24 h (scale bar, 100 mm; original magnification, 100). (E) Relative quantification of BMP9 adenovirus infection rate in C3H10T1/2 cells. (F) Western blotting shows BMP9 recombinant adenovirus affects the protein level of BMP9 at 24 h. (G) RT-qPCR assay showing BMP9 affects LGR4 expression. (H) Western blotting shows BMP9 affects the protein level of LGR4 at 24 h and 48 h. *P < 0.05, **P < 0.01; n Z 3.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Western Blot, Transfection, Infection, Recombinant, Quantitative RT-PCR, Expressing

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 3 Effect of LGR4 on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effects of BMP9 and/or LGR4 on RUNX2 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or LGR4 on RUNX2 protein level shown by Western blotting. (C) The effects of BMP9 and/or LGR4 on ALP activity shown by alkaline phosphatase assay. (D) The effects of BMP9 and/or LGR4 on OPN mRNA expression shown by RT-qPCR. (E) The effects of BMP9 and/or LGR4 on OPN level shown by Western blotting. (F) The effects of BMP9 and/or LGR4 on mineralization shown by alizarin red S staining. (G) The effects of BMP9 and/or LGR4 knockdown on RUNX2

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, ALP Assay, Staining, Knockdown

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 4 Effect of mTOR on BMP9-induced osteogenic markers in C3H10T1/2 cells. (A) The effect of BMP9 on Raptor shown by RT- qPCR. (B) The effects of BMP9 and/or Raptor knockdown on RUNX2 mRNA expression shown by RT-qPCR. (C) The effects of BMP9 and/or Raptor knockdown on RUNX2 protein level shown by Western blotting. (D) The effects of BMP9 and/or Raptor knockdown on OPN mRNA level shown by RT-qPCR. (E) The effects of BMP9 and/or Raptor knockdown on OPN protein level shown by Western blotting. (F) The effects of BMP9 and/or Raptor knockdown on mineralization shown by alizarin red S staining. (G) The effect of BMP9 on Rictor mRNA level shown by RT-qPCR. (H) The effects of BMP9 and/or Rictor on RUNX2 mRNA level shown by RT-qPCR. (I) The effects of BMP9 and/or Rictor knockdown on RUNX2 protein level shown by Western blotting. (J) The effects of BMP9 and/or Rictor knockdown on OPN mRNA level shown by RT-qPCR. (K) The effects of BMP9 and/or Rictor knockdown on OPN protein level shown by Western blotting. (L) The effects of BMP9 and/or Rictor knockdown on mineralization shown by alizarin red S staining. *P < 0.05, **P < 0.01; n Z 3.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Staining

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 5 Effect of LGR4 and/or Raptor knockdown on osteoblastic and adipogenic markers induced by BMP9 in C3H10T1/2 cells. (A) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 mRNA level shown by RT-qPCR. (B) The effects of LGR4 and/or Raptor knockdown on BMP9-induced RUNX2 shown by Western blotting. (C) The effects of LGR4 and/or Raptor knockdown on BMP9-induced ALP activity shown by histochemical staining. (D) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN mRNA level shown by RT-qPCR. (E) The effects of LGR4 and/or Raptor knockdown on BMP9-induced OPN level shown by Western blotting. (F) The effects of LGR4 and/or Raptor knockdown on BMP9-induced matrix mineralization shown by histo- chemical staining. (G) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg mRNA expression shown by RT-qPCR. (H) The effects of LGR4 and/or Raptor knockdown on BMP9-induced PPARg protein level shown by Western blotting. (I) The effects of LGR4 and/or Raptor knockdown on BMP9-induced lipid droplets formation shown by oil red O staining. *P < 0.05, **P < 0.01; n Z 3.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Activity Assay, Staining, Expressing

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 6 Effect of LGR4 and Raptor on the BMP9-induced skull defect repair. (A) The effect of LGR4 and Raptor knockdown on the bone formation induced by BMP9 (the area circled in red is the area of the defect site) shown by reconstruction of micro-CT analysis. (BeD) The effects of LGR4 and Raptor knockdown on bone formation induced by BMP9 indicated by quantitative results of the micro-CT assay of BV/TV, Tb.N, and BS/TV. BV/TV, bone volume per tissue volume; Tb.N, trabecular number; BS/TV, bone volume per bone surface area. *P < 0.05, **P < 0.01 vs. AdGFP control group; #P < 0.05, ##P < 0.01 vs. AdBMP9 group; DP < 0.05, DDP < 0.01 vs. AdBMP9 þ AdsiRaptor group; n Z 6.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Knockdown, Micro-CT, Control

Journal: Genes & diseases

Article Title: BMP9 induces osteogenic differentiation through up-regulating LGR4 via the mTORC1/Stat3 pathway in mesenchymal stem cells.

doi: 10.1016/j.gendis.2023.101075

Figure Lengend Snippet: Figure 7 Effect of mTORC1/Stat3 signal and LGR4 on BMP9-induced osteogenesis in C3H10T1/2 cells. (A) The effects of BMP9 and/or Raptor knockdown on LGR4 mRNA expression shown by RT-qPCR. (B) The effects of BMP9 and/or Raptor knockdown on LGR4 protein level shown by Western blotting. (C) The effects of BMP9 and/or Raptor knockdown on total and phosphorylated Stat3 levels shown by Western blotting. (D) The effects of AG490 and/or BMP9 on LGR4 mRNA shown by RT-qPCR. (E) The effects of AG490 and/or BMP9 on LGR4 shown by Western blotting. (F) The effects of AG490 and/or LGR4 on BMP9-induced mineralization shown by histochemical staining. (G) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 mRNA expression shown by RT-qPCR. (H) The effects of AG490 and/or LGR4 on BMP9-induced RUNX2 shown by Western blotting. (I) The effects of AG490 and/or LGR4 on BMP9- induced ALP activity shown by histochemical staining. (J) The effects of AG490 and/or LGR4 on BMP9-induced OPN mRNA expression shown by RT-qPCR. (K) The effects of AG490 and/or LGR4 on BMP9-induced OPN shown by Western blotting. AG490: Jak2/Stat3 inhibitor. *P < 0.05, **P < 0.01; n Z 3.

Article Snippet: Primary antibodies against BMP9 (sc-514211), LGR4 (sc7974), Runt-related transcription factor 2 (RUNX2; sc390715), osteopontin (OPN; sc-21742), phosphorylated signal sensor and activator of transcription 3 (p-Stat3, Tyr705; sc-8059), and b-actin (sc-47724) were purchased from Santa Cruz Biotechnology (Shanghai, China); antibodies against Rictor (27248-1-AP), Raptor (20984-1- AP), and PPARg (16643-1-AP) were bought from Proteintech (Wuhan, China); antibody against Stat3 (A1192) was purchased from ABclonal Biotechnology (Wuhan, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Staining, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains *

doi: 10.1074/jbc.M115.680009

Figure Lengend Snippet: Release of mature growth factor from BMP9·prodomain complex. Release of mature BMP9 domain after binding of an anti-pro-domain antibody was confirmed by application of BMP9·pro-domain to the pro-domain ELISA using anti-pro-domain antibodies AF3879 as capture and BAF3879 as detection (A). B, supernatant from A and recombinant mature BMP9 standard were applied to the mature BMP9 ELISA using anti-mature BMP9 antibodies MAB3209 as a capture and BAF3209 as detection. The mature BMP9 ELISA detected both mature BMP9 standard and released mature BMP9 from pro-domain ELISA supernatant.

Article Snippet: Antibodies used included anti-mature BMP9 antibodies MAB3209 and its biotinylated conjugate BAF3209 and anti-pro-domain antibodies AF3879 and its biotinylated conjugate BAF3879 and were purchased from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant