Figure 2.

Senescent satellite cells and mesenchymal progenitor cells were present in DMD rats. (a–c) Quantification of mRNA levels of senescence markers (p16, p19, and p21) in WT and DMD rat TA muscles (n = 6, each). (d) In situ hybridisation of CDKN2A mRNA using RNAscope on TA muscle sections from 6-month-old WT and DMD rats. Shown are representative images of tissues from WT (top) and DMD rats (middle) with CDKN2A mRNA appearing as brown dots. Scale bar = 50 μm. A higher magnification image from the dotted area is shown in the bottom frame. Scale bar = 10 μm. The white arrowheads show the mononucleated CDKN2A mRNA⁺ cells. (e) Skeletal muscle primary cells from 6-month-old DMD rats were subjected to CDKN2A mRNA in situ hybridisation using RNAscope before the immunocytochemistry of Pax7, CSPG4, CD45, and CD31. Scale bar = 10 μm. White arrowheads indicate DAB signal. White arrows indicate CD45⁺ or CD31⁺ cells. (f) Quantification of CDKN2A⁺ Pax7⁺ cells per all Pax7⁺ cells (shown as SC) and CDKN2A⁺ CSPG4⁺ cells per all CSPG4⁺ cells (shown as MPC) in skeletal muscle primary cells from DMD rats (n = 4, each). Data are expressed as mean + SEM, and were compared by Tukey Kramer’s test. For (a–c), the result of statistical comparison only between the genotypes at each indicated ages was displayed. When a significant age-related difference was observed by the Tukey–Kramer’s test, the † mark was added beside the legend of the graph. *p < 0.05. **p < 0.01. ***p < 0.001.