Figure 7.

TG2 promotes fusion of C2C12 myoblasts without using its crosslinking activity. (A) mRNA expression levels of Table 2. in differentiating C2C12 myoblasts in the presence or absence of 50 µM ZDON, a conformation inhibitor of TG2 crosslinking activity, added at day 0 of differentiation (B) Biotin-cadaverine incorporation into proteins of differentiating C2C12 myoblasts in the presence and absence of 100 µM monodansylcadaverine, a competitive inhibitor of TG2 crosslinking activity, detected by Western blot analysis. Protein loadings on the Coomassie dye-stained gel are also shown. (C) Representative light- and fluorescent microscopic images of C2C12 myoblasts differentiated for 6 days in the absence or presence of 50 µM ZDON added at day 0 or day 4 of differentiation, or in the presence of 100 µM monodansylcadaverine added at day 0 of differentiation. MYHC4 was visualized by using anti-MYHC4 antibody (green) and nuclei by DAPI (blue). Scale bars, 100 µm. (D) Fusion index of C2C12 cells differentiated for 6 days in the absence and presence of TG2 inhibitors. (E) The length of myotubes generated from C2C12 myoblasts differentiated for 6 days in the absence or presence of TG2 inhibitors (F) Alterations in the number of viable C2C12 cells grown in growth and differentiation medium in the presence or absence of 50 µM ZDON added at day 0 determined by PrestoBlue staining. (G) mRNA expression levels of myogenic genes in differentiating C2C12 myoblasts in the presence or absence of 50 µM ZDON (added at 0 day of differentiation) determined by qRT-PCR. (H) Protein expression levels of myosin heavy chain 4 (MYHC4) in differentiating C2C12 myoblasts in the presence or absence of 50 µM ZDON (added at day 0 of differentiation) determined by Western blot analysis. β-actin was used as a loading control. One representative blot of three is shown. All the data are expressed as mean ± SD of three independent experiments. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, Student’s t-test).