Figure 4: P40/50 and P50/70 differ in myogenic marker expression after prolonged cultivation.

(a) P40/50 and P50/70 cells were passaged at day 4 and cultured in growth medium for another 5 days. Subsequently, immunofluorescence staining was performed to visualize Pax7; cell nuclei were stained with DAPI. Pax7 was localized in the nuclei, and a higher proportion of nuclei was positive for the transcription factor in P50/70 cells, in contrast to P40/50 cells. Percentages of positive cells of each sample (n = 5) are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q₁–Q₃), and the resulting outliers are included as circles. For statistical analysis, Mann-Whitney Rank Sum Test was performed, *p ≤ 0.05. (b) Flow cytometric analysis of proliferating SC from SM or LD muscle after 8 days of cultivation. P40/50 cells show a significantly higher proportion of cells positive for Pax7. Percentages of positive cells of each sample are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q₁–Q₃), and the resulting outliers are included as circles. For statistical analysis, Students t-test (Desmin, MyoG, MyHC) or Mann-Whitney Rank Sum Test (Pax7) was performed, *p ≤ 0.05, n = 4 (MyoG, MyHC), 5 (Pax7) and 9 (Desmin).