Fig. 5

A ROS levels in whole embryos (top, P = 0.3463) and CD41^lo HSPCs (bottom, P = 0.0323) measured by flow cytometry for CellROX Orange^TM ROS-dye at 72 hpf. MFI is displayed relative to the average of control for each experiment [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 6/condition, 14 clutches]. B Representative WISH for runx1/cmyb expression at 72 hpf in control and bnip3lb morphants (NAC Tx: 10 µM, 48–72 hpf). Scale bar, 100 µM. C Phenotypic distribution of runx1/cmyb expression scored in embryos from (B) (NAC Tx: 10 µM, 48–72 hpf; P = 0.0001) [Pearson’s chi-square (two-sided): n = 23/condition, 4 clutches]. D Frequency of CD41^lo HSPCs in Tg(−6.0itga2b:EGFP) embryos by flow cytometry at 72 hpf after splice-targeted bnip3lb MO knockdown (*P = 0.0458), NAC (*P = 0.0333; Tx: 10 µM, 48–72 hpf) or both (P = 0.7929) vs controls; NAC vs both (*P = 0.0311). [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 4 clutches]. E EdU analysis in whole disaggregated control and bnip3lb morphants by flow cytometry at 72 hpf (P = 0.7613) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 14 clutches]. F EdU analysis within CD41^lo HSPCs of control and bnip3lb morphants by flow cytometry at 72 hpf (*P = 0.0248) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 14 clutches]. G qPCR for apoptotic marker genes at 72 hpf after bnip3lb knockdown [significance for full apoptotic panel: two-way ANOVA, *P = 0.0001); individual significance for casp2 (*P = 0.0236) and noxa1 (*P = 0.0426) expression, two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 15/condition, 7 clutches]. H Confocal imaging of HSPCs at 72 hpf in the CHT of control and bnip3lb morphants, with or without NAC treatment (Tx: 10 µM, 48–72 hpf). Kdrl+ vasculature (blue), acridine orange apoptosis (green), Runx1+ HSPCs (magenta); dual-labeled apoptotic Runx1+ cells indicated with yellow arrows. Scale bar, 50 µM. I Proportion of apoptotic HSPCs marked with acridine orange in control compared to NAC (P = 0.4613), bnip3lb-MO (*P = 0.0472) and bnip3lb-MO + NAC (P = 0.2679) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 3/condition]. A, C–G, I Source data are provided as a Source Data file.