Fig. 4. Overexpression of murine Hes6 (mHes6) and Hes6DBM (mHes6DBM) in C2C12 myoblasts decreases the number of cells undergoing irreversible cell cycle withdrawal. (A) C2C12 cells were cultured in DM for 120 hours, fixed and stained for immunofluorescent analysis of the proportion of nuclei in Troponin-T-positive cells, as shown in Fig. 3. Results of a typical experiment are shown. The mean of five random fields containing a total of at least 600 nuclei is shown, error bars indicate s.d. *P=0.004, **P=0.001, comparing murine Hes6 and Hes6DBM, respectively, with ß-gal control using a two-tailed unpaired t-test. There was no significant difference between murine Hes6 and Hes6DBM. (B) C2C12 cells were cultured in DM for 48 or 120 hours, fixed and stained for immunofluorescent analysis of the proportion of p21^Cip1-positive nuclei as shown in Fig. 3. The mean proportion of p21^Cip1-positive nuclei expressed as a percentage of the total number nuclei in three independent experiments is shown. At least 1000 nuclei for each cell type were counted from random microscope fields at each time point in each experiment. Error bars show s.e.m. *P=0.037, **P=0.040, comparing murine Hes6 with ß-gal control using a two-tailed paired t-test at 48 and 120 hours, respectively. ⁺P=0.013, ⁺⁺P=0.009, comparing murine Hes6DBM with ß-gal control using a two-tailed paired t-test at 48 and 120 hours, respectively. There was no significant difference between the percentage of p21^Cip1-positive cells for murine Hes6 and Hes6DBM at either time point. (C) Cells were cultured in DM for 5 days and then placed in GM containing 50 µM BrdU for 20-22 hours. Cells were then fixed and stained for BrdU to determine the proportion of BrdU-positive nuclei as shown in Fig. 3. The mean proportion of BrdU-positive nuclei expressed as a percentage of the total number nuclei in three independent experiments is shown. At least 1000 nuclei for each cell type were counted from random microscope fields at each time point in each experiment. Error bars show s.e.m. *P=0.008, **P=0.015, comparing murine Hes6 and Hes6DBM, respectively, with ß-gal control using a two-tailed paired t-test. There was no significant difference between murine Hes6 and Hes6DBM.

Return to article