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

Figure 3.

Figure 3

Transcriptional regulation of Tfeb by androgen receptor and oestrogen receptor beta signalling. (A) Relative mRNA expression of Tfeb in C2C12 cells treated with indicated inhibitors for 12 h. HF, hormone‐free media; F, Flutamide, an Ar inhibitor; M, MPP, an Esr1 inhibitor; P, PHTPP, an Esr2 inhibitor. (B) Representative immunoblot (top) of Tfeb and relative band densities (bottom) in C2C12 cells treated with the inhibitors for 24 h. (C) Relative luciferase units (RLU) of CLEAR motif sequence in C2C12 cells expressing 4× CLEAR treated with the inhibitors for 24 h. (D) Putative binding sites of Ar in the Tfeb gene. TSS, transcription start site; ORF, open reading frame. (E) Chromatin‐immunoprecipitation analysis of MuSCs from skeletal muscles of 4‐month‐old mice with control IgG and anti‐Ar antibody. (F) mRNA expressions of Tfeb target‐genes in C2C12 cell treated with DHT (right y axis) or 17β‐estradiol (left y axis) for 24 h. (G–K) Three‐month‐old WT and dKO mice were orally administered with tamoxifen for 5 days, and then, MuSCs were isolated from hindlimb muscles 6 months after tamoxifen treatment. MuSCs transduced with lentiviral vectors containing control or Tfeb [Lv‐Cont‐IRES‐tdTomato (Lv‐Cont) or Lv‐Tfeb‐IRES‐tdTomato (Lv‐Tfeb)]. MuSCs were analysed 96 h after Tfeb transduction. Immunostaining for LC3 (G) and quantification of MFI (H) with or without Baf treatment for 6 h. Relative mRNA expression of senescence‐associated genes (I). (J and K) Sixteen hours after transduction, equal numbers of transduced MuSCs were transplanted into young injured Pax7 CreER ;ROSA‐DTA mice pre‐treated with tamoxifen for 4 weeks before injury. Ten days after MuSC transplantation, TA muscles were analysed. IHC images for eMyHC and tdTomato (J), and quantification of mean CSA of tdT+eMyHC+ myofibres (K). Comparisons by one‐way ANOVA with Tukey's post hoc test (A–C, F, and H) and unpaired t‐test (E). Scales: 10 (G) and 100 μm (J) bars, mean ± SEM; n = 3–4 independent experiments; *P < 0.05, **P < 0.01, n.s. not significant. dKO, double knockout; MuSCs, muscle stem cells.