Figure 5.

Chronic oxidative conditions and/or upregulation of S100B expression increase BMP-7 secretion and autocrine pro-brown adipogenic effects of BMP-7. (a) C2C12 myoblasts were cultured in GM for 72 h in the absence or presence of 100 μM PQ or transfected with S100b. Parallel L6C11 and L6C8 myoblasts were cultured in GM for 72 h. Culture media were collected, TCA precipitated and analyzed for expression of BMP-7 by western blotting. (b) C2C12 myoblasts were cultured in GM for 72 h in the absence or presence of 100 μM PQ±non-immune IgG (control and PQ) or neutralizing anti-BMP-7 antibody (4 μg/ml). Cells were stained with ORO. (c) Conditions were as in b except that cells were analyzed by real-time PCR. (d) C2C12 myoblasts were transfected with S100b in GM and treated with non-immune IgG or neutralizing anti-BMP-7 antibody (4 μg/ml). Cells were analyzed by real-time PCR. (e) L6C11 myoblasts were cultured for 72 h in the absence (left panel) or presence (middle panel) of L6C8 conditioned medium and stained with ORO. Parallel L6C8 myoblasts (right panel) also were stained with ORO. (f) Conditions were as in (e) except that cells were analyzed by real-time PCR. (a,b,e): one representative experiment of three. The fold changes (±S.E.M.) (N=3) were determined (c, d, f). *, ** and ***significantly different from control (P<0.05, P<0.01 and P<0.001, respectively). Bars in (b, e)=100 μm