Fig. 6

Atf4a regulates mitochondrial homeostasis through Parkin-mediated mitophagy in zebrafish. (A) Protein level of Atg7, Atg4b, P62 and Lc3a/b in wt and atf4a−/− zebrafish embryos treated with normoxia or hypoxia at 72 hpf. β-actin was used as a loading control. (B, C) Relative expression levels of parkin and pink1 mRNA in wt and atf4a−/− zebrafish treated with normoxia and hypoxia at 72 hpf. (D) Zebrafish parkin promoter sequence, extending 1000 bp upstream of the transcription start site (TSS). The red arrow indicates the TSS. Predicted CREB/ATF-binding sites (Genomatix matrices) and the consensus ATF4-binding site (bold) are shown. Lower panels: Schematic diagrams of parkin dual luciferase reporter constructs with wild-type (wt), 1×, 2×, and 3× predicted ATF4-binding site sequences. (E) Relative luciferase activity in wt and atf4a−/− embryos microinjected with the indicated parkin luciferase reporter plasmids and renilla luciferase reporter plasmid. (F) Relative mtDNA levels in wt, atf4a−/− and atf4a−/− microinjected with parkin or pink1 mRNA. (G) Viability of wt, atf4a−/− and atf4a−/− microinjected with parkin or pink1 mRNA under hypoxic conditions. 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 < 0.0001. parkin: Parkin RBR E3 ubiquitin protein ligase; pink1: Pten induced kinase 1; β-actin: Beta-actin.