Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression
Figure 4
Smad3 and TGF-β alter the levels of gene expression in C2C12 cells.
C2C12 cell populations infected with retrovirus without the myc-Smad3 gene (Empty Vector, EV) or retrovirus encoding wild-type Smad3 were treated with either 1 µM SB431542 or 100 pM TGF-β for 24 hours before RNA isolation. The 16 genes were amplified by RT-PCR using the primer sets shown in Table S3. The annealing temperature and number of cycles of PCR were optimized for each primer set. The overexpression of Smad3 had modest effects on the levels of some genes (Adamts4, Cst6, Pdgfb, Pai1 and Wnt11) but had detectable effects on the levels of other genes (Fermt1, Gfpt2, Il11, Mmp9, Myl1, Npy, Olfm2, Sgcg). The expression levels of three genes, Cst6, Myl1 and Sgcg, were decreased by TGF-βtreatment. Overexpression of Smad3 caused further reductions in the levels of Myl1 and Sgcg.