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Fig. 1
a Western blot analysis was performed to detect the expression of LC3-I and LC3-II in intact (sham control) and regenerating hearts from 1 to 14 days. The protein from three hearts was loaded into each lane, and GAPDH was used as the loading control. b, c Autophagic flux assay with a western blot of LC3 was conducted (b). The expression of LC3-II was quantified. The data are presented as mean ± SD, n = 3 repeats, **P < 0.01 vs cryoinjured hearts with CQ treatment (c). d, e The sham (control) or cryoinjured hearts of Tg(cmv:GFP-LC3) fish were isolated, embedded in paraffin, sectioned, and dual-immunostained with an anti-GFP antibody to detect LC3 (in green) and an anti-MF20 antibody to detect the cardiomyocytes (in red), after which they were co-stained with DAPI to label the nuclei (in blue). IA: injured area; V: ventricle. The arrowheads show the GFP-LC3 puncta in cardiomyocytes. Scale bars: 50 µm (d). The number of GFP-LC3 puncta in sham and in the injured area at 1 dpc, 4 dpc, 7 dpc, and 14 dpc was quantified. The data are presented as mean ± SD, n = 3 hearts, **P < 0.01 vs sham (e). f, g Electron micrographs of an intact (sham) heart, and a regenerating heart in the IA at 1 dpc and 7 dpc. In the injured area, there was an accumulation of autophagic vacuoles (see blue arrowheads). M: mitochondria. Scale bars: 2 µm (f). The number of autophagic vacuoles in sham and the injured area at 1 dpc and 7 dpc was quantified. The data are presented as mean ± SD, n = 3 hearts, **P < 0.01 vs sham (g).