Figure 6: Link between Mkx and TGFβ and BMP signalling pathway component during ligament differentiation. | Nature Communications

Figure 6: Link between Mkx and TGFβ and BMP signalling pathway component during ligament differentiation.

From: Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs

Figure 6

(a,c) qRT–PCR analyses of the expression of TGFβ/Smad- and BMP/Smad-related genes in C3H10T1/2-V and C3H10T1/2-VM cells. mRNA levels in C3H10T1/2-V cells were normalized to 1. Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001. Statistical differences were assessed with Student’s t-test. (b,d) Western blot analyses of C3H10T1/2-V and C3H10T1/2-VM cells. TGFβ-dependent phosphorylation of Smads is shown by anti-p-Smad2/3 antibodies. BMP-dependent phosphorylation of Smads is shown by anti-p-Smad1/5/8 antibodies. (e) Luciferase assay to determine responsiveness to BMP2. Id1-Bre luciferase was transfected into C3H10T1/-V and C3H10T1/2-VM cells, followed by treatment with or without BMP2 (100 ng ml−1). Fold-induction values in the presence of BMP2 compared with the basal levels of each cell line are indicated. **P<0.01. Statistical differences were assessed with Student’s t-test. (f–k) C3H10T1/2-V and C3H10T1/2-VM cells were subjected to chondrocyte (f,h,j) and osteocyte (g,i,k) differentiation. (f) Alcian blue staining of C3H10T1/2-V and C3H10T1/2-VM cells after induction of chondrocyte differentiation. (g) Alizarin red staining of C3H10T1/2-V and C3H10T1/2-VM cells after induction of osteocyte differentiation. Scale bars, 200 μm. (h) Mucopolysaccaride quantification after chondrogenic induction. Error bars represent s.e.m. *P<0.05. Statistical differences were assessed with Student’s t-test. (i) Calcification evaluation after osteogenic induction. Error bars represent s.e.m. ***P<0.001. Statistical differences were assessed with Student’s t-test. (j,k) qRT–PCR analyses of mesodermal gene expression in C3H10T1/2-V and C3H10T1/2-VM cells in the presence or absence of induction with BMP2. mRNA levels in C3H10T1/2-V cells without induction were normalized to 1. Error bars represent s.e.m. **P<0.01, ***P<0.001. Statistical differences were assessed with Student’s t-test. BMP, bone morphogenetic protein; TGFβ, transforming growth factor β.

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