Figure 1.

SIX1 is functional in C2C12 myoblasts. (A) Western blot quantitation of SIX1 expression in differentiating C2C12 cells. Total cell lysates were used, and membranes were probed for SIX1 and myogenin expression. T0 h, confluent myoblasts at the onset of differentiation induction. T24 h, myoblasts induced to differentiate for 24 h. Beta-tubulin is given as a loading control. (B) Binding of SIX1 to the endogenous myogenin promoter, measured by qChIP. The chromatin from C2C12 cells at different stages of differentiation was immunoprecipitated with antibodies against SIX1 or MYOG, or control rabbit IgG. Enrichment of the ChIP samples for the myogenin promoter sequence and a negative control region (Hoxd10), were measured by qPCR. The binding signal expressed as a proportion of the starting material recovered in the ChIP reaction (percent of input). Asterisks indicate that the enrichment passes three significance tests, as indicated in ‘Materials and Methods’ section. The data represent the average of at least three biological replicates. Error bars: standard error on the mean (SEM). (C) Venn diagram showing the overlap in regions bound by SIX1 in myoblasts, at 24 h of differentiation, and in MT. The numbers given are the number of bound regions, and those in parentheses represent the number of distinct genes bound. (D) Validation of SIX1 ChIP-on-chip findings using qChIP. ChIP and data analysis were performed as described in Figure 1B. Average of at least three biological replicates. Error bars: SEM. Asterisks indicates significant binding event, as in B.