Fig. 4.

Reduction of Dkk3 level rescued age-related muscle atrophy. a Adenovirus-encoding shRNA against Dkk3 was injected to TA muscles in old mice (20 months) intramuscularly. Adenovirus-encoding scramble shRNA was injected to the TA muscle in a mouse with the same age and gender as control. Both pieces of shRNA were co-expressed with GFP to label the infected muscle fibers in vivo. Injections were performed once a day for 7 continuous days. TA muscles were harvested at day 14 for further analysis. b Gene expression levels of Dkk3, Fbxo32, and Trim63 in TA muscles injected with shRNA against Dkk3 or scramble control as indicated by RT-qPCR. Error bars indicated s.d. and were based on five independent experiments. ** indicated p < 0.01. c Representative immunofluorescent staining images of muscle cross sections derived from TA muscles in old mice treated with shRNA against Dkk3 or scramble control. The cross areas of all 1500 fibers in each TA muscle were checked. Red indicated laminin staining; green indicated GFP; DAPI indicated nuclei; merge indicated merged images of laminin, GFP, and DAPI staining. Scale bars, 50 μm. d Percentage distribution of muscle fiber cross section area derived from muscles treated with shRNA against Dkk3 or scramble control. Black dots indicated muscle sections with scramble control treatment. Gray squares indicated muscle sections with Dkk3 RNAi. e The percentage of TA muscle (by weight) in whole body muscles derived from mice treated with shRNA against Dkk3 or scramble control. Error bars indicated s.d. and were based on five independent experiments. ** indicated p < 0.01. f Specific titanic force of TA muscles treated with shRNA against Dkk3 or scramble control in old mice (20 months). The specific titanic force of TA muscles in old mice were compared to that of young mice (3 months). g 1/2 relaxation time of TA muscles treated with shRNA against Dkk3 or scramble control in old mice (20 months). 1/2 relaxiation time of TA muscles in old mice were compared to that of young mice (3 months). All error bars indicated s.d. based on 5 independent experiments. ** indicated p < 0.01. * indicated p < 0.1. NS indicated no significant changes. All p-values were based on two-tailed t-test