24 hpf zebrafish embryos were treated with A 30 Gy IR-irradiation, B 5 μM Thapsigargin (Thaps.), and C 3.3 μM Phorbol 12-myristate 13-acetate (PMA); and qRT-PCR was performed at 6 h (A) or 4 h (B, C) after treatment. Expression levels were normalized to GAPDH. n = 9 (A, B) and n = 7 (C) from ~30 pooled embryos per sample. Bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fold change (FC) is indicated.

A Schematic of Puma wild-type and mutant proteins. (BH3, BH3 domain). B Schematic of Noxa wild-type and mutant proteins. C Schematic of p53 wild-type and mutant proteins. TAD transactivation domain, DNA-binding DNA-binding domain, NLS nuclear location signal, OD Oligomerization domain, BR basic region. D Schematic of Mdm2 wild-type and mutant proteins (p53 binding, p53-binding domain; Acidic, acidic domain; ZF, zinc finger domain; RF, ring finger domain). E Schematic of p63 wild-type and mutant proteins (CT, C-terminal region; p63 mutant transcripts undergo nonsense-mediated decay, NMD). F Schematic of p73 wild-type and mutants. Arrow points out the target site of zinc finger, TALEN, or CRISPR-Cas9 gene editing. Orange indicates the key domain in each protein. Light green bar labels full-length wild-type or in-frame truncated mutant protein and the length of amino acid (aa) sequence is indicated. Red tail indicates out-of-frame part of the truncated protein. The figure is created with BioRender.com.

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.

A Anti-active Caspase-3 staining on 12, 15, 18, and 21 hpf mdm2^+/+ and mdm2^−/− zebrafish embryos. B qRT-PCR analysis of puma and noxa in 15 hpf mdm2^+/+, mdm2 siblings (mdm2+/+ and mdm2^+/−) and mdm2^−/− zebrafish embryos. n = 3 from ~30 pooled embryos per sample. Bars represent mean ± SEM. **p < 0.01. C Anti-active Caspase-3 staining on 12, 15, 18, and 21 hpf mdm2^+/+ bbc3^−/− and mdm2^−/− bbc3^−/− zebrafish embryos. D Gross images of 15, 18, and 21 hpf mdm2^+/+, mdm2^−/−, and mdm2^−/− bbc3^−/− zebrafish embryos.

A Anti-active Caspase-3 (The Upper Panel) and TUNEL (The Lower Panel) staining on 28 hpf (4 h post treatment) wild-type zebrafish embryos with DMSO alone or with DMSO plus 5 μM Thaps. Representative figures showing phenotypic categories of the apoptotic severity. Arrows points out apoptotic area in tail region in WT embryos with DMSO alone or with DMSO plus 5 μM Thaps for 4 h. Scale bar, 1000 μM. B Quantification of the fluorescence intensity of tail region of DMSO-treated and Thaps. treated embryos in mild and severe categories with anti-active Caspase-3 staining. Each dot represents mean fluorescence intensity (MFI) of the tail region of individual embryos from three independent experiments. Bars represent mean ± SEM. **p < 0.01.****p < 0.0001. C Quantification of the fluorescence intensity of tail region of DMSO-treated and Thaps. treated embryos in mild and severe categories for TUNEL staining. Each dot represents mean fluorescence intensity (MFI) of the tail region of individual embryos from two independent experiments. Bars represent mean ± SEM. ****p < 0.0001. D Loss of puma (not noxa) partially rescued Thaps. induced apoptosis at 24 hpf. Ratio of phenotypic categories in wild type, bbc3^−/− and pmaip1^−/− zebrafish embryos. n = 7 (wild type and bbc3^−/−) and n = 4 (pmaip1^−/−) from pooled embryos per sample. The total number of Thaps. treated embryos: wild type > 1000, bbc3^−/− > 900 and pmaip1^−/− > 550. Bars represent mean ± SEM. ****p < 0.0001. E qRT-PCR analysis of puma and noxa after DMSO or 5 μM Thapsigargin treatment in 24 hpf zebrafish embryos across time (2hpt and 4hpt). Expression levels were normalized to GAPDH. n = 5 (26-hpf WT with or w/o 2 h post Thaps. treatment) and n = 9 (28-hpf WT with or w/o 4 h post Thaps. treatment) from ~30 pooled embryos per sample. Bars represent mean ± SEM. ***p < 0.001; ****p < 0.0001.

A Loss of p63 partially rescued Thaps. induced apoptosis at 24 hpf. Ratio of phenotypic categories of the apoptotic severity in wild type, tp63^−/−, sibling tp63^+/− and sibling tp63^+/+ zebrafish embryos at 4 h after DMSO or Thaps. treatment. Embryos from the intercross of heterozygous-mutant tp63 adults were genotyped after sorted based on the apoptotic severity at tail region. B Loss of p53 and p73 cannot rescue Thaps. induced apoptosis at 24 hpf. Ratio of phenotypic categories in 24 hpf wild-type and tp53^−/− tp73^−/− zebrafish embryos at 4h after DMSO or Thaps. treatment. n = 6 (A) and n = 4 (B) from pooled embryos per sample. The total number of Thaps treated embryos: wild type > 660, tp63^−/− > 360, tp63^+/− > 700, tp63^+/+ > 320 and tp53^−/− tp73^−/− > 420. Bars represent mean ± SEM. ****p < 0.0001.

A Anti-active Caspase-3 (The upper panel) and TUNEL (The Lower Panel) staining on 28 hpf (4 h post treatment) wild-type zebrafish embryos with DMSO or DMSO plus 3.3 μM PMA. Representative figures showing phenotypic categories of the apoptotic degree. Arrows points out apoptotic area in tail region in WT embryos with DMSO alone or with DMSO plus 3.3 μM PMA for 4 h. Scale bar, 1000 μM. B Quantification of fluorescence intensity of tail region of DMSO-treated and PMA-treated embryos in mild and severe categories with anti-active Caspase-3. Each dot represents MFI of the tail region of individual embryos from three independent experiments. Bars represent mean ± SEM. ****p < 0.0001. C Quantification of the fluorescence intensity of tail region of DMSO-treated and PMA-treated embryos in mild and severe categories for TUNEL staining. Each dot represents mean fluorescence intensity (MFI) of the tail region of individual embryos from two independent experiments. Bars represent mean ± SEM. ****p < 0.0001. D Loss of both puma and noxa partially rescued PMA-induced apoptosis at 24 hpf. Percentage of phenotypic categories in wild type, bbc3^−/− and pmaip1^−/− zebrafish embryos. n = 4 (wild type and bbc3^−/−) and n = 7 (pmaip1^−/−) from pooled embryos per sample. The total number of PMA-treated embryos: wild type > 600, bbc3^−/− > 440, and pmaip1^−/− > 800. Bars represent mean ± SEM. ***p < 0.001. E qRT-PCR analysis of puma and noxa after DMSO or 3.3 μM PMA treatment in 24 hpf zebrafish embryos across time (2hpt and 4 hpt). Expression levels were normalized to GAPDH. n = 5 (26 hpf WT) and n = 7 (28 hpf WT) from around 30 pooled embryos per sample. Bars represent mean ± SEM. ***p < 0.001; ****p < 0.0001.

Percentage of phenotypic apoptotic categories in wild type, tp63−/−, sibling tp63+/−, and sibling tp63+/+ zebrafish embryos at 4 h after treatment with DMSO or PMA. B Percentage of phenotypic categories in 24 hpf wild type and tp53−/− tp73−/− zebrafish embryos at 4 h after DMSO or PMA treatment. n = 6 (A) and n = 5 (B) from pooled embryos per sample. The total number of Thaps treated embryos: wild type > 600, tp63−/− > 140, tp63+/− > 390, tp63+/+ > 180, and tp53−/− tp73−/− > 370. Bars represent mean ± SEM. ****p < 0.0001.