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. 2020 Nov 27;12(1):177–191. doi: 10.1002/jcsm.12653

Figure 5.

Figure 5

Prevention of Antide‐induced muscle stem cell (MuSC) senescence by sex hormone administration, not by Notch signalling activation. (A–F) Three‐month‐old WT and Pax7 CreER ;ROSA‐N1 (N1SC/OE) transgenic mice were orally administered with Tmx for five consecutive days and treated with Antide for 10 months as indicated. Note that GFP in MuSCs from Pax7 CreER ;ROSA‐N1 (N1SC/OE) mice is coexpressed with the intracellular domain of Notch1 after successful recombination by Cre recombinase. A scheme for MuSC‐specific activation of Notch signalling (top) and Antide administration (bottom) (A). Flow cytometry analysis of MuSCs isolated from Antide‐treated WT or N1SC/OE mice. Note that GFP+ and GFP− cells are N1ICD‐overexpressing and normal MuSCs, respectively, in one mouse (B). Relative mRNA expression of Notch target genes and senescence‐associated genes in GFP+ and GFP− MuSCs isolated from Antide‐treated N1SC/OE mice (C). Quantification of single‐cell electrophoretic assay (D). Veh‐treated or Antide‐treated WT mice were used as control group (Cont). Note that a distinct head and tail by single cell electroporation mean intact DNA and broken pieces of damaged DNA, respectively. 46 Representative images for γH2AX and GFP in freshly isolated MuSCs (E) and quantification (F). (G–L) A scheme for Antide and DHT cotreatment. Silastic tubes containing Veh or DHT were implanted into 3‐month‐old C57BL/6 mice, and they were treated with Antide 6 mg/kg/week for 10 months (G). Relative mRNA expression of senescence‐associated genes in MuSCs from Sham/Veh, Sham/Antide or DHT/Antide‐treated mice (H). BaCl2 injury was induced in TA muscles of indicated mice. At 3 dpi, representative images (I) and relative percentages of Pax7+Ki67+ cells (J). Arrows and arrowheads indicate Pax7+Ki67+ and Pax7+Ki67− cells, respectively. IHC images for laminin (K) and (L) quantification of CSA of TA muscles at 21 dpi. Scales: 100 (H) and 50 μm (E). Comparisons by one‐way analysis of variance with Tukey's post hoc test and paired t‐test. Bars, mean ± SEM; 4 animals per group and K; n = 7 (Sham/Veh), 4 (Sham/Antide), and 5 (DHT/Antide) TA muscles from 4 animals per group; *P < 0.05, **P < 0.01, n.s. not significant.