Fig. 3

Atf4a deficiency induces mitochondrial damage in zebrafish. (A) Relative mtDNA levels in wt and atf4a−/− zebrafish treated with normoxia and hypoxia at 72 hpf. The mtDNA levels were measured by RT-qPCR and normalized by nucleus DNA levels. (B, C) Relative expression level of 12SrRNA, aco2, atp5a1, and atp5b mRNA in wt and atf4a−/− zebrafish treated with normoxia and hypoxia at 72 hpf. mRNA levels were determined by RT-qPCR and normalized to β-actin mRNA. (D) Tetramethylrhodamine methyl ester (TMRM) fluorescence intensity in wt and atf4a−/− zebrafish embryos at 72 hpf, measured by flow cytometry. (E) Relative mtDNA levels in wt, atf4a−/−, or atf4a−/− microinjected with atf4a mRNA at 72 hpf. (F) Relative mtDNA levels in wt and atf4a−/− at 24, 26, 28, 30, 32, 34, and 36 hpf. (G) Transmission electron microscopy (TEM) images of wt and atf4a−/− embryos treated with normoxia or hypoxia. Nucleus (purple), normal mitochondria (green), abnormal mitochondria (yellow), lysosome (arrows). Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001. aco2: Aconitase 2; atp5a1: ATP synthase F1 subunit alpha 1; atp5b: ATP synthase F1 subunit beta; β-actin: Beta-actin.