Research Paper Volume 13, Issue 8 pp 11336—11351

COX-2 promotes the osteogenic potential of BMP9 through TGF-β1/p38 signaling in mesenchymal stem cells

The effect of TGF-β1 and/or COX-2 on osteoblastic markers induced by BMP9 in C3H10T1/2 cells. (A) Western blotting shows that NS-398 and/or BMP9 affect Smad2/3 and p-Smad2/3 (phosphorylated Smad2/3). (B) Quantification results of Western blots assay shows Smad2/3 or p-Smad2/3 was affected by NS-398 and/or BMP9. (C) Western blotting shows the level of Smad2/3 or p-Smad2/3 was affected by COX-2 and/or BMP9. (D) Quantification results of western blot assay shows the level of Smad2/3 and/or p-Smad2/3 was affected by COX-2 and/or BMP9. (E) ALP staining shows the effect of NS-398, TGF-β1, and/or BMP9 on ALP activity. (F) Quantification of ALP staining shows that ALP activities were affected by NS-398, TGF-β1, and/or BMP9. (G) Alizarin Red S staining shows that the mineralization was affected by NS-398, TGF-β1, and/or BMP9. (H) Quantification results of Alizarin Red S assay shows that mineralization was affected by NS-398, BMP9, and/or TGF-β1. (I) ALP assay shows the BMP9-induced ALP activities was affected by COX-2 and LY364947. (J) Quantification of ALP assay shows that ALP activities were affected by COX-2, LY364947, and/or BMP9. (K) Alizarin Red S staining shows that the mineralization was affected by COX-2, LY364947, and/or BMP9. (L) Quantification results of Alizarin Red S staining shows that mineralization was affected by COX-2, LY364947, and/or BMP9. LY364947: TGF-βRI inhibitor; NS-398: COX-2 inhibitor. “**”p “#”p “##”p

Figure 3. The effect of TGF-β1 and/or COX-2 on osteoblastic markers induced by BMP9 in C3H10T1/2 cells. (A) Western blotting shows that NS-398 and/or BMP9 affect Smad2/3 and p-Smad2/3 (phosphorylated Smad2/3). (B) Quantification results of Western blots assay shows Smad2/3 or p-Smad2/3 was affected by NS-398 and/or BMP9. (C) Western blotting shows the level of Smad2/3 or p-Smad2/3 was affected by COX-2 and/or BMP9. (D) Quantification results of western blot assay shows the level of Smad2/3 and/or p-Smad2/3 was affected by COX-2 and/or BMP9. (E) ALP staining shows the effect of NS-398, TGF-β1, and/or BMP9 on ALP activity. (F) Quantification of ALP staining shows that ALP activities were affected by NS-398, TGF-β1, and/or BMP9. (G) Alizarin Red S staining shows that the mineralization was affected by NS-398, TGF-β1, and/or BMP9. (H) Quantification results of Alizarin Red S assay shows that mineralization was affected by NS-398, BMP9, and/or TGF-β1. (I) ALP assay shows the BMP9-induced ALP activities was affected by COX-2 and LY364947. (J) Quantification of ALP assay shows that ALP activities were affected by COX-2, LY364947, and/or BMP9. (K) Alizarin Red S staining shows that the mineralization was affected by COX-2, LY364947, and/or BMP9. (L) Quantification results of Alizarin Red S staining shows that mineralization was affected by COX-2, LY364947, and/or BMP9. LY364947: TGF-βRI inhibitor; NS-398: COX-2 inhibitor. “**”p < 0.01, “#”p < 0.05, and “##”p < 0.01.