Knockdown of MyoD dramatically reduces the expression of Cdc6 in growth-stimulated quiescent myoblasts. (A) C2C12 myoblasts were separately transduced with lentiviruses expressing two distinct shRNAs specific for MyoD (shMyoD-64 and -66) and a scramble shRNA (shCntrl). After 42 h, the myoblasts were brought to quiescence and then stimulated to grow for the indicated times in GM. (B and C) After stimulation, the myoblasts were harvested, and the expression of MyoD or Cdc6 was then monitored either by RT-PCR (B) or by Western blotting (C). NS denotes a nonspecific band. EV, empty vector. (D and E) Cross-linked chromatin was isolated from growth-stimulated quiescent myoblasts expressing a scramble shRNA (shCntrl) or an shRNA specific for MyoD (shMyoD-66). Afterward, the protein–DNA fragments were immunoprecipitated in parallel with anti-E2F3a or anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 site within the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA, which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out.