|
Fig. 6 Atrogin-1 is a modifier of and contributes to the pathogenesis of Duchenne muscular dystrophy. (A) Representative Western blot image for BiP, and total protein direct blue stain, on whole cell protein lysates obtained from 2 dpf and 4 dpf dmd+/+ or dmd–/– larvae. (B) Quantification of BiP levels normalized to total protein with 4 dpf dmd–/– larvae displaying a significant increase compared with dmd+/+ larvae, as determined using a 2-way ANOVA with Šidák’s multiple correction post hoc test. Data are shown as mean ± SD. (C–F) Representative birefringence images of 4 dpf dmd+/+; atrogin-1+/+ wild-type larvae and dmd–/– and atrogin-1–/– single and double mutants. Scale bar: 500 μm. (G) Quantification of normalized birefringence intensity, which is the mean birefringence intensity relative to area, in 4 dpf dmd+/+; atrogin-1+/+ wild-type larvae and dmd–/– and atrogin-1–/– single and double mutants, analyzed using a 1-way ANOVA with Tukey’s multiple correction post hoc test. Data are shown as mean ± SD. (H) Average speed, in mm/s, of dmd+/+; atrogin-1+/+ wild-type larvae and dmd–/– and atrogin-1–/– single and double mutants, as analyzed using as a 1-way ANOVA with Tukey’s multiple correction post hoc test. Data are shown as mean ± SEM for 3–4 biological replicates. (I and J) Live images of 4 dpf larvae expressing GFP RNA or atrogin-1-IRES-GFP RNA. Scale bar: 200 μm. (K–N) Birefringence images of 4 dpf dmd+/+ and dmd–/– larvae injected with GFP RNA or atrogin-1-IRES-GFP RNA. Scale bar: 500 μm. (O) Quantification of normalized birefringence intensity, which is the mean birefringence intensity relative to area, in 4 dpf dmd+/+ or dmd–/– larvae injected with GFP RNA or atrogin-1-IRES-GFP RNA, as analyzed using a 2-way ANOVA with Šidák’s multiple correction post hoc test. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments performed in triplicate with the total number of fish examined in each replicate being documented in Supplemental Table 2.