Restoration of autophagic flux via rapamycin attenuates ALLCinduced cellular dysfunction and cell death in vivo A, B Immunoblot analysis of LC3II and p62 expression in zebrafish lysate, 3 days postinjection of ConLC or ALLC (100 µg/ml). GAPDH was used as a loading control. N = 5 (A); N = 4 (B). *P = 0.043, ^#P = 0.029. C. Transmission electron micrographs of cardiac tissue of Con-LC and AL-LC-injected zebrafish 3 days postinjection (5 days postfertilization). Top panels show changes in mitochondrial morphology and presence of doublemembrane structures (autophagosomes) at 6,800× in AL-LC fish compared to Con-LC fish. Bottom panel shows higher magnification of mitochondrial structure and autophagosomes. Scale bar = 500 nm. N = 3 per group. D. Zebrafish were treated with 10 nM rapamycin after receiving Con-LC or AL-LC injection. Immunoblot analysis was performed to measure p62 expression and normalized to GAPDH as a loading control. Fifteen fish were homogenized per experiment. Quantitative analysis is shown for comparison below representative blots. N = 3. *P = 0.011. E. Peak flow was measured in zebrafish embryos using color Doppler echocardiography. Representative color Doppler peak flow tracings of zebrafish 3 days postinjection with or without treatment with rapamycin. Peak flow quantitation analysis is shown in the panel below. N = 5. *P = 0.001. F. Cell death was quantified in zebrafish 3 days post-injection with or without rapamycin treatment. Expression of active caspase 3 was measured using immunoblot. 15 zebrafish were homogenized per experiment. N = 4. *P= 0.029. G. Representative confocal images of hearts isolated from zebrafish 3 days post-injection with or without rapamycin treatment. Hearts are stained for TUNELpositive nuclei (red) and DAPI counterstain. TUNEL-positive nuclei are quantified and graphed in the right panel. Scale bar = 25 µm. N = 4 per group. *P = 6.7 × 10⁴, ^#P = 0.001. H. Kaplan–Meier analysis of zebrafish survival following injection of Con-LC or AL-LC in the absence or presence of rapamycin. Survival was monitored daily. The survival of AL-LC-injected fish was significantly prolonged in the presence of rapamycin. N = 25 per group.*P = 0.0001.