Figure 2. Assessments of AR-reporter in the fish tail vs head reveal differential roles of zKeap1a and zKeap1b.

Homozygous Tg(gstp1:GFP) fish were crossed with wt fish. Resulting heterozygous embryos were injected with the stated morpholino (MO) at the 1- to four-cell stage. (See experimental Workflow in Appendix 1-Scheme 1). Image quantitation was performed on the head/tail-regions as illustrated. Note: GFP expression was detected using immunofluorescence (IF) in fixed fish, analyzed by red fluorescence. The IF protocol is used because auto-fluorescence in the green channel is high in fish and prevents accurate quantitation and IF avoids concerns regarding effects of electrophile on GFP fluorescence. ATG MOs used for single-MO injection, SPL MOs used for simultaneously knocking down zKeap1a and zKeap1b; see Figure 1—figure supplements 3–5 and Figure 2—figure supplements 1–2 for MO validations and Appendix for MO sequences. Also see Figure 1—figure supplements 1–5, Figure 2—figure supplements 1–2, Figure 3—figure supplement 1. (A) Quantitation of GFP expression (which indicates relative basal AR-levels) in the tail (left panel) and head (right) of Tg(gstp1:GFP) zebrafish larvae following MO-knockdown of the indicated zKeap1 and zNrf2 paralogs. No. embryos analyzed: Control MO (38), zNrf2a MO (32), zNrf2b MO (21), zKeap1a MO (21), zKeap1b MO (22), zKeap1a and zKeap1b MOs (29). (B) Quantitation of the relative fold change of AR level (GFP signal) in the tail (left panel) and head (right) following bulk electrophile (NE; see Figure 1A inset) exposure. No. embryos analyzed: Control MO (48), zNrf2a MO (27), zNrf2b MO (27), zKeap1a MO (24), zKeap1b MO (29), zKeap1a and zKeap1b MOs (20). All numerical data present mean ± sem. Numbers above the bars represent analysis by two-tailed t-tests.

Figure 2—figure supplement 1. Validations of zKeap1a-ATG-MO.

zKeap1a-mRNA rescues the effects of MO knockdown. (Also see Figure 1—figure supplements 3B and 4,5). (A) Representative images of wildtype (WT) and Tg(gstp1:GFP) zebrafish at 8 hpf. (B) Representative images of zebrafish (co-)injected with the indicated MO and mRNA (2 nl of 0.2 mM zKeap1a-ATG-MO; 250 ng/μL Halo-TEV-zKeap1a mRNA), treated at 4 hpf with 10 μM NE (4 hr treatment period followed by image acquisition at 8 hpf). (C) GFP expression from the experiments in (B) was quantified using the measure tool of Image-J(NIH). Yolk sac was excluded from quantification (see sketches on top). Sample sizes analyzed: (left panel, DMSO-treated): Control MO, n=26; zKeap1a MO, n=18; zKeap1a MO + zKeap1 a mRNA, n=16; (right panel, NE-treated): Control MO, n=20; zKeap1 MO, n=35; zKeap1a MO + zKeap1 a mRNA, n=26. Fish age: 8 hpf. Scale bars, 500 μm. All numerical data present mean ± sem. Numbers above the bars represent analysis by two-tailed t-tests.

Figure 2—figure supplement 2. Validations of zKeap1b-ATG-MO.

zKeap1b-mRNA rescues the effects of MO knockdown. (Also see Figure 1—figure supplements 3B, 4 and 5,). (A) Quantitation of GFP expression in the tail (left panel) and head (right panel) of zebrafish (co-)injected with the indicated MO and mRNA (2 nl of 0.25 mM zKeap1b-ATG-MO; 250 ng/μL Halo-TEV-zKeap1b mRNA). Sample sizes analyzed: Control MO, n=42; zKeap1b MO, n=36; zKeap1a MO + zKeap1 a mRNA, n=40. (B) Quantitation of the relative fold change of AR level (GFP signal) in the tail (left panel) and head (right panel) following bulk NE exposure at 30 hpf (20 μM, 4 h treatment period followed, by image acquisition at 36 hpf). Embryos were (co-)injected with MO and mRNA at 1–4 cell stage (2 nL of 0.25 mM zKeap1b-ATG-MO and 250 ng/μL Halo-TEV-zKeap1b mRNA). Sample sizes analyzed: Control MO, n=42; zKeap1b MO, n=39; zKeap1a MO + zKeap1 a mRNA, n=32. Fish age: 34 hpf. All numerical data present mean ± sem. Numbers above the bars represent analysis by two-tailed t-tests.