Loss of puma not noxa rescues IR-induced apoptosis in p53-dependent manner.A Anti-active Caspase-3 staining on 30 hpf (1 hpi and 6 hpi panel) or 48 hpf (24 hpi panel) embryos. 29 hpf wild-type, pmaip1−/−, tp53−/−, tp63−/−, tp73−/− and bbc3−/− zebrafish embryos were treated with 30 Gy IR-irradiation and fixed at 1 hour post treatment (1 hpi panel); 24 hpf embryos were treated with 30 Gy IR-irradiation and fixed at 6 h (6 hpi panel) or 24 h (24 hpi panel) after treatment. Arrows in WT points out active apoptotic area in head region in WT embryos for 6 hpi and 24 hpi. Scale bar, 1000 μM. B Anti-active Caspase-3 staining of 30 hpf (6 hpi) wild-type, pmaip1−/−, tp53−/−, tp63−/−, tp73−/−, and bbc3−/− zebrafish embryos treated w/o IR (non-IR) and with 15, 30, 45, 60, and 100 Gy IR. Scale bar, 1000 μM. C p53 protein expression level after IR-induction. Western blot analysis was performed using protein extracts from 30 hpf (6 hpi) wild type, tp53−/−, bbc3−/−, and pmaip1−/− zebrafish embryos with (30 Gy) or without IR treatment. D qRT-PCR analysis of puma and noxa after IR-irradiation in zebrafish embryos. 24 hpf wild-type or tp53−/− zebrafish embryos were treated with 30 Gy IR-irradiation and RNA samples for qRT-PCR were harvested at 6 h after IR-irradiation. Expression levels were normalized to GAPDH. n = 6 from ~30 pooled embryos per sample. Bars represent mean ± SEM. ****p < 0.0001.
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