Fig. 2

a, b Neutrophil levels were tracked over time following Keap1-hydroxynonenylation by Z-REX (red squares) [against untreated control (blue dots)] in Tg(lyz:TagRFP) expressing either Halo-TEV-Keap1 (a) or Halo-P2A-Keap1 (b). Halo-P2A-Keap1 control construct (Supplementary Fig. 1a inset) cannot undergo Keap1-hydroxynonenylation via Z-REX. P values calculated with Student’s two-tailed t-test. c Whole-mount immunofluorescence (IF) staining of active Caspase-3 (the key apoptotic executioner Caspase) and neutrophils in Tg(lyz:TagRFP) embryos following Keap1-hydroxynonenylation by Z-REX. Scale bars, 500 μm. Box in Merge panel marks the area magnified. White arrows mark colocalizations. d Equation used to calculate fold change (shown in inset panels). This value takes into account the starting number of immune cells and allows a fair comparison between different sets. The value is independent of starting immune-cell count and development. e Keap1-hydroxynonenylation with Z-REX was performed in Tg(lyz:TagRFP) embryos co-injected with control MOs, or MOs targeting Nrf2a or Nrf2b (500 μM). (ATG-MO: an MO targeting the translation start site; SPL-MO: an MO inhibiting splicing). Inset at right: analysis of fold change (Z-REX/non-treated) in neutrophil count. See Supplementary Fig. 5. P values were calculated with ANOVA and Tukey’s multiple comparisons test. f Treatment of Tg(lyz:TagRFP) embryos with two inhibitors of Caspase-3 [AZ-10417808 (reversible, non-peptide) and Z-DEVD-FMK (covalent)] ablated Z-REX promoted neutrophil loss. Inset at right: analysis of fold change (Z-REX/non-treated) in neutrophil count. P values in black were calculated with two-tailed unpaired Student’s t-test; P values in blue were calculated with ANOVA and Dunnett’s multiple comparisons test. g Treatment of Tg(lyz:TagRFP) embryos with Calpain inhibitor I had no effect on Z-REX-promoted neutrophil loss. Inset at right: analysis of fold change (Z-REX/corresponding non-Z-REX-condition) in neutrophil count. P values were calculated with two-tailed unpaired Student’s t-test. h Treatment of Tg(lyz:TagRFP) embryos with Bcb suppressed Z-REX-promoted neutrophil loss. Inset at right: analysis of fold change (Z-REX/corresponding non-Z-REX-condition) in neutrophil count. P values were calculated with two-tailed unpaired Student’s t-test. All data present mean ± SEM. All sample sizes are listed in Supplementary Methods. Source data are provided as a Source data file.