a, AMPKα1/α2 deficiency reduced phospho-AMPKα T172 and phospho-ACC levels and increased phospho-S6 levels, as expected. Each lane contained protein from 30,000 sorted cells. +/+ indicates AMPKα1/α2fl/fl cells and −/− indicates Mx1-Cre; AMPKα1/α2fl/fl cells 6 days after pIpC treatment. b, Lkb1 or AMPKα deletion did not significantly affect DCFDA staining (ROS levels) in HSCs (b, c), MPPs or WBM (c) cells 11 days after pIpC treatment. d, NAC treatment for two weeks did not rescue the depletion of Lkb1-deficient HSCs (*, p<0.05 by Student’s t-test). e, Mitochondrial DNA copy number was significantly reduced 6 days after Lkb1 or AMPKα deletion (*, p<0.05; **, p<0.005 in all panels). f, g, Mitochondrial mass significantly increased 11 days after Lkb1 deletion in HSCs, and MPPs, but not in WBM cells. AMPKα deletion significantly increased mitochondrial mass in all populations 11 days after pIpC treatment. A representative histogram shows Mitotracker staining in HSCs after Lkb1 or AMPKα deletion (f). h, ATP levels were significantly reduced in HSCs after Lkb1 or AMPKα deletion, 6 or 11 days after pIpC treatment. i, j, Mitochondrial membrane potential (Δψ) was significantly reduced after Lkb1 deletion in HSCs (i, j) and MPPs but not in WBM cells (j) or GMPs (k) 11 days after pIpC treatment. AMPKα deletion did not reduce Δψ in any cell population (i, j). l, AMPKα deletion did not cause transient expansion or rapid depletion of HSCs, but did modestly reduce HSC frequency 70 days after pIpC treatment (p<0.05). m, AMPKα-deficient HSCs were capable of long-term multilineage reconstitution 6 days after pIpC treatment, in contrast to Lkb1-deficient HSCs (). All data (mean±standard deviation) are from 3 to 7 independent experiments.