Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2022 Jan 1.

Published in final edited form as: Mater Sci Eng C Mater Biol Appl. 2020 Nov 27;120:111748. doi: 10.1016/j.msec.2020.111748

Figure 2. — (I) BMP-9 triggered Smad signaling pathway is resistant to Noggin in Mesenchymal Stem Cells (MSCs). (A) Noggin inhibition of Smad activation in BMP 2. Adenoviral (Ad) expressing Noggin (Ad-Noggin) or Ad-Red Fluorescent Protein (RFP) were exposed to the C2C12 cells; cells were cultured overnight in Fetal Bovine Serum (1%) overnight. Three different media were categorized as cmBMP2 (a, b), cmBMP9 (c, d), and cmGFP (Green Fluorescent Protein) (e, f) in which the infected cells were treated for 2 h. Immunofluorescence staining using anti-Smad 1/5/8 antibody (from Santa Cruz Biotechnology); Negative control used for the experiment was either control IgG or absence of primary antibody staining. (B) Smad 6/7, induced by the BMP-9 signaling cascade, is expressed in the presence of Noggin; proves the resistance of BMP-9 towards Noggin inhibition. Sub confluent C2C12 cells were used for this experiment; these cells were infected with Ad-BMP 2/9 and Ad-Noggin or Ad-RFP for 30 h. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using mouse Smad 6 and Smad 7 primers was done on the isolated total RNA from the infected cells. Normalization control used was GAPDH with triplicates done for all assays. Noggin vs. RFP expression (Smad 5 and Smad 7) ** p value < 0.01, # p value < 0.05. (II) Noggin resistance is displayed by BMP-9 during ectopic bone formation. (A). Ectopic bone masses formed with two different cell lines, C2C12 and mouse embryonic fibroblasts (MEFs), infected with the Ad-BMP-9 and then injected subcutaneously injected into athymic mice with and without Noggin. These samples were from 4 weeks post-injection. (B) Histological evaluation of the samples harvested from Ad-BMP-9 infected C2C12 cell injection site; (a) H& E staining, (b) Trichrome staining, and (c) Alcian blue staining. (C) Histological evaluation of the samples harvested from Ad-BMP-9 infected MEFs cell injection site; (a) H& E staining, (b) Trichrome staining, and (c) Alcian blue staining. X200 magnification. BM – Bone matrix; CM – chondroid or cartilage matrix. Figures 2. (I) and (II) reprinted from [45] with permission from John Wiley and Sons. Copyright © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Figure 2. — (I) BMP-9 triggered Smad signaling pathway is resistant to Noggin in Mesenchymal Stem Cells (MSCs). (A) Noggin inhibition of Smad activation in BMP 2. Adenoviral (Ad) expressing Noggin (Ad-Noggin) or Ad-Red Fluorescent Protein (RFP) were exposed to the C2C12 cells; cells were cultured overnight in Fetal Bovine Serum (1%) overnight. Three different media were categorized as cmBMP2 (a, b), cmBMP9 (c, d), and cmGFP (Green Fluorescent Protein) (e, f) in which the infected cells were treated for 2 h. Immunofluorescence staining using anti-Smad 1/5/8 antibody (from Santa Cruz Biotechnology); Negative control used for the experiment was either control IgG or absence of primary antibody staining. (B) Smad 6/7, induced by the BMP-9 signaling cascade, is expressed in the presence of Noggin; proves the resistance of BMP-9 towards Noggin inhibition. Sub confluent C2C12 cells were used for this experiment; these cells were infected with Ad-BMP 2/9 and Ad-Noggin or Ad-RFP for 30 h. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using mouse Smad 6 and Smad 7 primers was done on the isolated total RNA from the infected cells. Normalization control used was GAPDH with triplicates done for all assays. Noggin vs. RFP expression (Smad 5 and Smad 7) ** p value < 0.01, # p value < 0.05. (II) Noggin resistance is displayed by BMP-9 during ectopic bone formation. (A). Ectopic bone masses formed with two different cell lines, C2C12 and mouse embryonic fibroblasts (MEFs), infected with the Ad-BMP-9 and then injected subcutaneously injected into athymic mice with and without Noggin. These samples were from 4 weeks post-injection. (B) Histological evaluation of the samples harvested from Ad-BMP-9 infected C2C12 cell injection site; (a) H& E staining, (b) Trichrome staining, and (c) Alcian blue staining. (C) Histological evaluation of the samples harvested from Ad-BMP-9 infected MEFs cell injection site; (a) H& E staining, (b) Trichrome staining, and (c) Alcian blue staining. X200 magnification. BM – Bone matrix; CM – chondroid or cartilage matrix. Figures 2. (I) and (II) reprinted from [45] with permission from John Wiley and Sons. Copyright © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.