Fig. 2. Inhibition of MMP activity in the elongation stage blocks myotube formation without affecting the genetic program. (A) Differentiating C2C12 cells were treated with BB94 (10 µM) during the indicated time period, and 0.1% of DMSO was added throughout the cell culture duration. (B) The total cell number, the numbers of myogenin-positive cells and the fusion rates were measured at 144 hours. The results are presented as the mean ± s.e.m. (n = 6). (C) Expression levels of differentiation marker genes were measured by semi-quantitative RT-PCR at 144 hours, including muscle creatine kinase (MCK) and embryonic myosin heavy chain (eMyHC). The results are normalized with G3PDH and presented as the mean ± s.e.m. (n = 3). (D) Anti-MHC immonostaining (green signal) with Hoechst counterstaining (blue signal) at 144 hours. Bar, 100 µm. (E) Luciferase reporter assay for representative myogenic transcriptional factors. Reporter constructs have tandem repeats of the DNA binding sites for the indicated myogenic transcription factors. 4RE houses four tandem binding sites for MRFs, 3MEF2 contains three tandem binding sites for MEF2 and 3SRE contains three tandem binding sites for SRF. The results are presented as the mean ± s.e.m. (n = 6).