ZAK? mediates menadione-induced apoptosis and death in zebrafish. (A) U2OS cells were treated with menadione (250 ?M) for the indicated times. Lysates were analyzed by immunoblotting with the indicated antibodies. (B) U2OS cells individually deleted for ZAK (?ZAK), ASK1 (?ASK1), and both (?ZAK/?ASK1) were treated with menadione (250 ?M) for the indicated times. Lysates were analyzed as in (A). (C) Model of ROS-induced activation of p38 and JNK kinases by parallel sensing mechanisms. ZAK? responds to ROS-induced impairment of ribosomal translation (left), whereas ASK1 activity is directly controlled by intracellular ROS levels (right). (D) Genomic location of guide RNA sequences (blue) and derived knockout alleles in exon 2 of zebrafish zak? (top) and zak? (bottom) genes. A 33-bp insertion in mutated zak? is highlighted in red, with the position of an in-frame STOP codon underlined and in bold. PAM, protospacer adjacent motif. (E) Genotyping of WT and CRISPR-modified alleles of zak? and zak? genes from (D). A single zebrafish larva per genotype was lysed and submitted to genomic PCR with the indicated primer pairs. Amplified bands were resolved by agarose gel electrophoresis. (F) Schematic of experiments with menadione treatment of zebrafish larvae. Zebrafish eggs were incubated in the presence of menadione (9 ?M) and/or NAC (60 ?M) from 6 hours postfertilization and up to 5 days. Larvae were scored as dead or alive once per day, and the number of fish with developmental phenotypes was determined. For detection of apoptosis, larvae treated for 3 days were fixed and subjected to TUNEL staining. (G) Thirty zebrafish larvae with each of the indicated genotypes were treated and scored as in (F). Only larvae with cardiac arrest were scored as dead. The data shown are from one representative experiment out of the three performed. dpf, days post fertilization. (H) Representative images of larvae from (G) at day 3. WT fish treated with menadione (Mena) presented with darkened yolk sac, cardiac edema, and short kinked tails (red arrows). The addition of NAC or deletion of zak? largely obliviated these pathologies. (I) WT and zak??/? larvae were treated as in (F) for 3 days, fixed, and subjected to TUNEL staining. Photographs show representative images of the tail part of the larvae. All scale bars, 500 ?m.
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