Analysis of the molecular consequence of ARNT deficiency; simultaneous deletion of Hif-1α and Hif-2α phenocopy of the Arnt KO phenotype. (A) Representative Western blot analysis for Phospho-S6 and total S6 as a loading control on FACS-sorted LSK cells (n = 3 ctrl, n = 3 KO). (B-C) Representative FACS plots of Phospho-S6 staining in control and KO LT-HSCs (B), quantified for all hematopoietic progenitors in (C), showing no difference in Phospho-S6 levels (n = 6 ctrl, n = 6 KO). (D) ATP assay performed on FACS-sorted MPPs and LT-HSCs (n = 6 ctrl, n = 6 KO). (E) qRT-PCR analysis of Arnt KO LT-HSCs (n = 5 ctrl, n = 6 KO). (F) VEGF enzyme-linked immunosorbent assay performed on BM and spleen extracts (n = 6 ctrl, n = 6 KO). (G) Vav-Cre;Hif-1α^fl/fl/Hif-2α^fl/fl DKO mice have fewer LT-HSCs than control mice (n = 3 ctrl, n = 3 DKO). (H) Annexin V staining revealed enhanced apoptosis in DKO LT-HSCs (n = 6 ctrl, n = 6 KO). (I) qRT-PCR analysis of sorted DKO LT-HSCs (n = 3 ctrl, n = 4 KO). *P < .05, **P < .01.