Mutations on Smad3 have differential effects on the TGF-β inducibility of six endogenous genes in C2C12 myoblasts.

Populations of C2C12 cells from two independent infections (A and B) with retroviruses expressing wild-type or mutant Smad3s were treated with 100 pM TGF-β1 (induced) or the TGF-β-receptor kinase inhibitor SB431542 (basal) for 24 hours. Quantitative-RT-PCR was used to quantify the levels of the six indicated gene products and these were normalized to the level of beta-actin mRNA in each lysate. The normalized levels for both the basal and induced cell lysates are shown in Figure 5 and Figure S1. TGF-β inducibility, or fold increase over basal level, was calculated as the ratio of the TGF-β-induced level divided by the basal level of expression. In the C2C12 cells infected with retrovirus expressing wild-type Smad3 (both the A and B infections), the fold increases over basal induced by TGF-β were: MMP9 6±1; Il-11 31±5; Tnfaip6 11±2; Fermt1 8±2; Olfm2 31±10; Wnt11 12±2. The TGF-β inducibility provided by the mutant Smad3 proteins was normalized to the TGF-β inducibility provided by wild-type Smad3 in the infected cells which was set to 100%. The standard deviations are indicated in parenthesis. *Variance in the very low basal expression levels were the primary contributors to the large standard deviation.