Fig. 3. Analysis of effects of overexpression of murine Hes6 and Hes6DBM on C2C12 myoblast differentiation. C2C12 myoblasts were infected with bicistronic retroviral vectors encoding ß galactosidase (ß-gal), murine Hes6 (mHes6) or Hes6DBM (mHes6DBM) and GFP as described in the Materials and Methods. Cells were cultured for 5 days in DM and then analysed by immunofluorescence. (A) Structure of retroviral vector. A bicistonic retroviral vector was used. Transcription of RNA encoding the insert, an internal ribosome entry site (IRES) and cDNA encoding EGFP is driven by the 5' long terminal repeats (LTR). Translation of the bicistronic RNA produces two proteins, the inserted protein and EGFP. Expression of the puromycin resistance gene (puro) is driven by the SV40 viral promoter (SV40). (B-D) EGFP expression. Unstained cells were examined for EGFP fluorescence to confirm translation of the retrovirally expressed bicistronic RNA. There was no apparent difference in the level of EGFP expression, but in cells overexpressing Hes6 or Hes6DBM, the myotubes were elongated and narrower than ß-gal-expressing cells. Appearances shown are typical of five independent experiments. (E-G) Troponin-T expression. Cells were stained with an anti-Troponin-T antibody. Myotubes expressed Troponin-T, a marker of terminal differentiation. The different morphology of myotubes in murine Hes6 and Hes6DBM cultures is seen; the number of nuclei per myotube is lower in murine Hes6 and Hes6DBM transduced cells compared with ß-gal expressing cells. Appearances shown are typical of five independent experiments. (H-J) p21^Cip1 expression. Cells were stained with an anti-p21^Cip1 antibody, disclosed with a Cy3-conjugated secondary antibody. Nuclei were stained with DAPI. Rows of p21^Cip1-positive nuclei correspond to multinucleate myotubes. Fewer p21^Cip1-positive nuclei occur in the murine Hes6- and HES6DBM-expressing cells (see Fig. 4). (K-M) BrdU labelling to detect cells capable of re-entering into the cell cycle. To determine the proportion of cells in each culture that had undergone irreversible cell cycle withdrawal, cells were exposed to GM containing 50 µM BrdU for 20-22 hours after 5 days in DM. Nuclei were then stained with an anti-BrdU antibody and disclosed with a Cy3-conjugated secondary antibody. Nuclei were then stained with DAPI. More BrdU-positive cells are found in the murine Hes6- and Hes6DBM-expressing cells (see Fig. 4).