Figure 4.

Low doses of UT did not change either the cell cycle or cellular senescence in murine cultured myoblasts. C₂C₁₂ cells were treated with a mixture of 25 µg/mL IS and 10 µg/mL PC (UT) or vehicle (CT) for 48 h. (a) Scratch wounding was made in the confluent monolayer and wound closure was monitored over the next 24 h with or without 10% FBS. Wounds were photographed at 0, 18, and 24 h after wounding to measure the degree of wound closure. A representative experiment shows only the photographs taken at 0 and 24 h after wounding. The graph represents the percentage of wound closure with respect to time 0 h. The results showed are mean ± SEM from five experiments. (b) C₂C₁₂ cells were treated for 48 h in culture medium with 10% FBS. Propidium iodide incorporation was assessed by flow cytometry. A typical experiment is shown. Results are the mean ± SEM from four experiments. (c) C₂C₁₂ cells were treated for 48 h with or without 10% FBS. PCNA expression was analysed by western blotting. A typical blot is shown. Full-length blot is presented in Supplementary Figure S3. The bar graph shows the densitometric analysis of the bands. The results, expressed as a percentage of control, are the mean ± SEM from seven different experiments. *p < 0.05 versus CT FBS 0%. (d) C₂C₁₂ cells were treated for 24, 48, or 72 h. Senescence-associated β-galactosidase activity was measured by confocal microscopy with the fluorogenic substrate C₁₂FDG. A typical experiment is shown. Results showed in the bar graph represent mean ± SEM from ten experiments. (e) C₂C₁₂ cells were treated for 48 h. Apoptosis was analysed using the Annexin V test via flow cytometry. A typical experiment is shown. Results are the mean ± SEM from six experiments.