Fig. 3.

Over-expression of Dkk3 in vivo induced muscle atrophy in young mice. a The schedule of virus injection. b Adenovirus-encoding Flag-Dkk3-IRES-GFP was injected to TA muscles in young mice (3 months) intramuscularly. Adenovirus-encoding GFP vector was injected to the TA muscle in mice with the same age and gender. Injections were performed once a day for 7 continuous days. TA muscles were harvested at day 14 for further analysis. A small portion of the TA muscles was used to make protein extracts. Anti-Flag immunoblotting was performed to detect the expression of the ectopic Flag-Dkk3. c Immunofluorescent staining images of muscle cross sections derived from TA muscles over-expressing Dkk3 or vector control. Red indicated laminin staining; green indicated GFP; DAPI indicated nuclei; merge indicated merged images of laminin, GFP, and DAPI. Scale bars, 50 μm. d Percentage distribution of muscle fiber cross section area derived from muscles over-expressing Dkk3 or GFP. The cross areas of all 1500 fibers in each TA muscle were checked. Black squares indicated muscle sections with ectopic Dkk3 expression. Gray dots indicated muscle sections expressing vector. Error bars indicated s.d. and were based on five independent experiments. e Fbxo32 and Trim63 mRNA levels were measured by RT-qPCR in TA muscles with ectopic Dkk3 or GFP expression. Error bars indicated s.d. and were based on five independent experiments. ** indicated p < 0.01. f Weight of TA muscles over-expressing Dkk3 was compared to that expressing vector control. Error bars indicated s.d. and were based on five independent experiments. ** indicated p < 0.01. fg Specific titanic force of TA muscles over-expressing Dkk3 or vector control in young mice (3 months). Error bars indicated s.d. and were based on 5 independent experiments. ** indicated p < 0.01. h 1/2 relaxation time of TA muscles over-expressing Dkk3 or vector control in young mice (3 months). 1/2 relaxation time of TA muscles in old mice were compared to that of young mice (3 months). Error bars indicated s.d. and were based on five independent experiments. ** indicated p < 0.01. All p-values were based on two-tailed t-test