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. 2019 Jun 4;33(8):9540–9550. doi: 10.1096/fj.201802298R

Figure 2.

Figure 2

emb-9 is degraded upon mitochondrial dysfunction caused by Antimycin A. A) Synchronized adult worms expressing emb-9::mCherry were treated with 0 (C, control), 2, 4, and 10 µM Antimycin A. After 36-h treatment with Antimycin A, animals were subjected to lysis, and Western blot was performed to analyze the amount of EMB-9 protein using anti-mCherry antibody. B) ImageJ was used to quantify the amount of EMB-9 protein, which was normalized to total tubulin content. Graph was prepared based on the values of the normalized amount of EMB-9; a significant decrease in EMB-9 was observed in 10 µM Antimycin A (3 biologic repeats). C) emb-9 TS mutant animals were synchronized and grown to young adulthood at 15°C (permissive temperature), after which the adult worms were either grown at 15°C or shifted to 25°C for up to 3 d. At D1, D2 and D3, animals were monitored for muscle damage using rhodamine phalloidin staining and confocal microscopy. A significant increase in muscle damage was observed in animals grown at the restrictive temperature at D1, D2, and D3 when compared with their respective permissive temperature conditions. The pattern of this muscle damage is illustrated in the representative images. Number of muscle cells/condition (n = 260, 196, 270, 312, 177, and 347 from left to right). Scale bars, 10 µM. D, E) Antimycin A–treated emb-9 OP worms were scored for worm paralysis and muscle damage as performed in Fig. 1A, B. Significant rescue of the paralysis phenotype was observed following 4 and 10 µM Antimycin A treatment, and muscle damage was recovered after 10 µM Antimycin A treatment. Twenty animals were analyzed for each experiment with 3 independent repetitions (n = 60 animals). Number of muscle cells/condition (n = 113, 106, 78, and 96 from left to right). The data for WT are the same as those used in Fig. 1A, B. They have been repeated here and in the following figures for comparison. OP, overexpression; pt, permissive temperature; rt, restrictive temperature. Letters (a–d) on top of bars indicate statistical significance; X, median value.