ROS produced by mitochondrial activity cause HSC expansion. (A) Treatment with the antioxidant NAC (10μM) reduced HSC formation and blocked the effect of glucose. Addition of exogenous hydrogen peroxide (0.05% H₂O₂) rescued HSC formation in OAA-treated embryos (n > 40/tx). (B) qPCR for runx1 expression confirmed the inhibitory impact of NAC exposure (ANOVA, P < .05, n = 3). (C) Quantitative analysis of double-positive (DP) cells in the AGM of cmyb:eGFP;lmo2:dsRed transgenic embryos by fluorescence microscopy confirms the inhibitory impact of NAC on HSC formation in vivo (t test, *P < .02 vs con, **P < .001 vs gluc, n = 5). (D-F) Morpholino knockdown of the endogenous metabolic antioxidant enzyme, peroxiredoxin (prdx1, 25μM) increased HSC formation as determined by (D) runx1/cmyb in situ hybridization, and observed in (E) runx1:eGFP and (F) CD41:eGFP transgenic zebrafish (n > 20/tx). (G) Quantification of runx1:eGFP and CD41:eGFP⁺ cells in prdx1 morphants by FACS revealed significant changes compared with controls (runx1: control 2.65% ± 1.38%; prdx1 MO 4.06% ± 1.02%, t test, *P = .023, n ≥ 9; CD41: control 0.22 ± 0.11%; prdx1 MO 0.533 ± 0.16%; t test, **P < .001, n = 10). (H) PF2 fluorescence intensity quantified by FACS in lmo2:dsRed endothelial cells revealed significantly increased ROS production after 1% glucose exposure (t test, *P < .0001, n = 4).