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Atrogin-1-dependent muscle dysfunctions are irreversible in chronically treated adult atrofish. (A) Brightfield imaging of adult zebrafish trunk after tissue-clearing showing degeneration of skeletal-muscle tissue in one-month-old atrofish treated with tamoxifen (TAM) for 5 months (bottom) compared to TAM-treated control fish (control; top) at 6 months of age. Arrows indicate regions of degeneration. (B) Quantification of the volume of muscle tissue in TAM-treated control fish (n = 6) or atrofish treated with either DMSO (n = 3) or TAM (n = 10). Statistical significance is determined by t-test, two-tailed, unpaired. (C) Representative images of 3D reconstructed trunk skeletal-muscle tissue after chronic TAM treatment in control fish (top) or atrofish (bottom) at 6 months of age. (D) Quantification of the volume expected after 3D reconstruction of atrofish trunk skeletal-muscle tissue (n = 10). Statistical significance is determined by t-test, two-tailed, paired. (E) Representative images of 3D reconstructed trunk skeletal-muscle tissue after chronic TAM treatment in atrofish at 6 months of age. (F, G) Quantification of the number of focal injuries in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction (F). Quantification of the average volume of injuries in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction (G). Statistical significance is determined by t-test, two-tailed, unpaired with Welch’s correction. (H) Quantification of the distribution of the injuries identified in (G) in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction. n in the graphic indicates the number of injuries per biological replicate. Injuries from 6 biological replicates for control or atrofish have been pulled together to determine statistical significance using t-test, two-tailed, unpaired. (I) Representative images of the numbers and sizes of injuries in trunk skeletal-muscle tissue after chronic TAM treatment in control (top) or atrofish (bottom) at 6 months of age. Each color represents a single injury, quantified in (H). (J) Quantification of swimming capacity of control fish and atrofish at 6 months of age treated with either DMSO or 4-OHT for 5 months (n = 19 for each condition). Statistical significance is determined by Mann-Whitney test, two-tailed, unpaired. (K) Quantification of swimming capacity of control fish (n = 9) and atrofish (n = 15) at 9 months of age, treated with TAM for 5 months, i.e., between 1 and 6 months of age, and then treated with either DMSO (ctrl, n = 5; atro, n = 4) or TAM (ctrl, n = 4; atro, n = 11) for an additional 3 months (i.e., until 9 months of age), to test for recovery of swimming function. Statistical significance is determined by a Two-way Anova. Error bars represent s.d. Scale bars: 1 mm.
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