Hif1α activity induced by elevated ROS mediates the effect of glucose metabolism on HSCs. (A) Exposure to CoCl₂ (500μM) or the hif1α prolyl hydroxylase inhibitor DMOG (500μM) expanded runx1/cmyb expression and rescued the impairment of HSC formation induced by antioxidants NAC and mitoQ (n > 50/tx). (B) qPCR demonstrated the effect of DMOG in enhancing runx1 expression and rescuing the effect of mitoQ exposure on HSCs (ANOVA, *P < .05, n = 3). (C) Fluorescent analysis of double-positive (DP) AGM cells in cmyb:eGFP;lmo2:dsRed transgenic embryos confirmed the positive impact of CoCl₂ on HSC formation in vivo that rescued the inhibitory effects of the antioxidants NAC and mitoQ (t test, *P < .01 vs con, **P < .001 vs CoCl_2, n = 5). (D) hif1a-MO knockdown decreased runx1/cmyb expression and blocked the effect of glucose on HSCs (43↓/55). (E) FACS quantification of runx1:eGFP⁺ cells following hif1a-MO knockdown revealed reduced HSC formation in the presence and absence of glucose (t test, *P < .001 vs con, **P < .001 vs gluc, n = 12). (F) Quantitative analysis of fluorescence microscopy images of cmyb:eGFP;lmo2:dsRed reporter embryos revealed the negative impact on HSCs of loss of hif1α function by morpholino knockdown could not be rescued by glucose exposure (t test, *P < .002 vs control, **P < .0001 vs glucose, n = 5).