Fig. 1. Stably expressing I-mfa siRNA in P19 cells initiate myogenesis. (A) Protein expression levels in different kinds of stable cell lines. The levels of endogenous I-mfa, ß-catenin and ß-tubulin and exogenous C-LEF-1-HA were determined by western-blot analysis. ß-tubulin was measured as an internal control. (B,C) P19 cell lines that stably express a control plasmid (Ctr), I-mfa siRNA (siRNA) or C-LEF-1, or coexpress C-LEF-1 and I-mfa siRNA were induced to differentiate after aggregation in the presence or absence of Wnt3a. At Day 9, cells were collected. MHC protein levels were analyzed via western blotting and quantified through the use of an Odyssey II quantification system as previously described (Pan et al., 2005) (B). Cells were also fixed and stained with DAPI (blue) and an anti-MHC antibody (green) (C). We established several individual clones for every kind of stable cell line. All the clones showed similar results, and we present only the results from one of them.