Fig. 2

Knockout Atf4a reduced hypoxia tolerance and impaired endoplasmic reticulum stress response in zebrafish. (A) Viability of wt (blue) and atf4a mutant (green) larvae under hypoxic conditions (n = 35 larvae per group). (B, C) Relative mRNA expression of gadd34 and igfbp1b in wt and atf4a−/− zebrafish embryos treated with normoxia or hypoxia at 72 hpf. mRNA levels were determined by RT-qPCR and normalized to β-actin mRNA. (D) Relative luciferase activity (HIF-1α transcriptional activity) in wt and atf4a−/− embryos injected with p2.1 and pRL-SV40 plasmids under normoxia and hypoxia. (E) Protein levels of p-PerkThr980, Perk, p-eIF2αSer51, eIF2α, and Bip in zebrafish embryos of wt and atf4a−/− treated with normoxia and hypoxia at 72 hpf. β-actin was used as a loading control. (F–I) Relative expression levels of perk, atf6a, chop, and xbp1s mRNA in wt and atf4a−/− zebrafish treated with normoxia and hypoxia at 72 hpf. (J) Viability of wt larvae pretreated with DMSO, ISRIB, or GSK2606414 under normoxia and hypoxia (n = 20 larvae per group). (K, L) Relative mRNA expression of gadd34 and igfbp1b in wt embryos pretreated with DMSO, ISRIB, or GSK2606414 under normoxia and hypoxia. The data represent the mean ± SEM of three independent experiments. Unpaired two-tailed Student's t-test was used for statistical analysis. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate a significant difference between the indicated groups. gadd34: Growth arrest and DNA damage—inducible protein 34; igfbp-1b: Insulin-like growth factor binding protein 1b; perk: Protein kinase R—like endoplasmic reticulum kinase; atf6a: Activating transcription factor 6 alpha; chop: C/EBP homologous protein; xbp1s: X-box binding protein 1 spliced form; β-actin: Beta-actin. ISRIB: Integrated stress response inhibitor; GSK2606414: PERK inhibitor.