FIGURE 1.

SelW expression is up-regulated in the early stage of C2C12 cell differentiation. A, phase-contrast microscopic images of proliferating and differentiating C2C12 cells cultured under the experimental conditions of this study. Proliferating C2C12 myoblasts were cultured in GM up to ∼50% confluence. To induce differentiation, C2C12 myoblasts were maintained in the same medium up to ∼100% confluence (D0). After exchanging the culture medium with DM, differentiating C2C12 cells were observed at 1, 3, and 5 days of differentiation (D1, D3, and D5). Bar, 100 μm. B, RT-PCR analysis of SelW and muscle-specific marker genes during C2C12 cell differentiation. Total RNA was isolated from C2C12 cells at the indicated time points and subjected to reverse transcription, followed by PCR with specific primers. Hprt1 was used as a loading control. A representative result of three independent experiments is shown. C, real time PCR analysis for the quantification of SelW mRNA expression during C2C12 cell differentiation. Using cDNA prepared in B, real time PCR was carried out with SelW- and Hprt1-specific primers. Expression level of SelW mRNA in proliferating C2C12 cells in GM was arbitrarily set at 1. Data are represented as means ± S.D. of four independent experiments performed in triplicate. D, expression of SelW and muscle-specific marker proteins during C2C12 cell differentiation. Western blotting was performed with cell lysates cultured at the indicated time points using specific antibodies and the generated polyclonal peptide antibody anti-SelW. α-Tubulin served as a loading control. The result shown is representative of three separate experiments. E, relative expression of SelW protein. The expression of SelW protein was quantified by scanning the SelW blots shown in D and was normalized based on the expression level of α-tubulin. Expression level of normalized SelW in proliferating C2C12 cells in GM was arbitrarily set at 1. Data are represented as means ± S.D. of three independent experiments.