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. 2016 Aug 3;7:12397. doi: 10.1038/ncomms12397

Figure 1. Mrf4 RNAi induces myofibre hypertrophy and protein synthesis in adult muscles.

Figure 1

(a) Rat soleus muscles co-transfected with GFP and LacZ or Mrf4 shRNAs and examined 14 days later. Scale bar, 100 μm. Cross-sectional area (CSA) of transfected fibres is shown below. Two hundred GFP-positive fibres from each muscle were analysed. (b) CSA of muscle fibres in innervated or 7 d denervated SOL and EDL muscles transfected with two different Mrf4 shRNAs (M1 and M2). Values normalized to fibres transfected with LacZ shRNAs in innervated muscles. Measures on 58 muscles, about 5 muscles per group, 15,485 fibres. (c) Mrf4 overexpression in adult SOL. CSA of muscle fibres transfected with Mrf4 cDNA normalized to control muscles (n=3). (d) Fibre size is increased in regenerating SOL co-transfected at day 3 after injury with GFP and LacZ or Mrf4 shRNAs, and examined 7 days later. Scale bar 100 μm. Right: CSA of regenerating fibres transfected with Mrf4 shRNAs (M1) normalized to control (n=3). (e) Fibre size is reduced by Mrf4 overexpression in regenerating muscle (n=3). (f) Rescue experiment for M1. The sequence recognized by M1 shRNA in rat Mrf4 transcripts (rMrf4) has a single base difference in human Mrf4 (hMrf4). hMrf4 is not silenced by M1 in HEK-293 cells (left), as shown by western blotting with anti-MRF4. Fibre growth induced by M1 in 14 days denervated SOL is prevented by M1-resistant hMrf4 (right, n=5). (g) Rescue experiment for M2. Same as in f, but using M2 shRNA specific for rat Mrf4 and M2-resistant mouse Mrf4 (mMrf4) (n=5). (h) Fibre size is not affected by myogenin knockdown. Left: HEK-293 cells transfected with myogenin cDNA (MyoG) and co-transfected with two shRNAs targeting myogenin (MG1 and MG2). GFP was co-transfected to determine transfection efficiency. Right: fibre size in unchanged by MG1 (n=3). (i) Increased Protein synthesis is increased in SOL by Mrf4 shRNAs (M1), as revealed by puromycin incorporation and immunostaining for puromycin-labelled peptides. Quantification is on the right (n=4). Data are presented as mean±s.e.m. from at least three independent experiments. Statistical analysis with Student's two-tailed t-test (*P<0.05, **P<0.01, ***P<0.001).