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AICAR and Metformin Treatment Improve the Muscle Phenotype in a Zebrafish Model of Dysferlin Deficiency (A) Morpholino oligonucleotides (MOs) targeting zebrafish dysferlin. (B) A single RT-PCR product of 301 bp was seen in 4-dpf embryos injected with 1.5 ng of control MO; two differently sized RT-PCR products of 301 and 195 bp were seen in 4-dpf embryos injected with 1.5 ng of dysferlin. (C) Panels represent the results of the birefringence assay (BR). The abnormal structure of the muscles is more clearly seen in dysferlin MO-injected fish (MO, lower panel) than in controls (CMO, upper panel) under BR. Scale bar, 200 μm. (D) Histogram of the percentage of normal, affected (reduced BR), and dead fish upon MO injection. After injecting 1.5 ng of MO, approximately 30.0% of the embryos exhibited reduced birefringence. The white, black, and gray bars show the percentage of normal, reduced BR, and dead fish, respectively. WT, uninjected fish; CMO, CMO-injected fish; MO, MO-injected fish. (E) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with AICAR for 4 days at 0, 0.1, 1.0, or 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. (F) AICAR treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 0.1, 1.0, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 31.1% were BR reduced. AICAR treatment at concentrations of 0.1, 1.0, and 10 mM reduced the percentage of BR-reduced fish to 22.2%, 17.8%, and 17.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05. (G) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with metformin for 4 days at 0, 1, and 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments and were normalized to those without metformin treatment. (H) Metformin treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 33.3% were BR-reduced fish. Metformin treatment at concentrations of 1 and 10 mM reduced the percentage of BR-reduced fish to 25.0% and 20.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05.
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