Extended Data Fig. 5. Properties of autophagy-deficient and old HSCs, and effect of ROS scavenging in Atg12^cKO mice.

a–c, Characteristics of Cnt and cKO HSCs: (a) apoptosis measured by cleaved caspase 3 (CC3) activity, (b) protein synthesis measured by O-propargyl-puromycin (OP-puro) IF staining, and (c) cycling activity measured by EdU incorporation. d, e, Characteristics of young and old HSCs: (d) ROS levels, and (e) protein synthesis with representative OP-puro IF staining (left) and quantification (right). f, Scheme for N-acetylcysteine (NAC) in vitro treatment and representative example of colony formation in methylcellulose from NAC-treated Cnt and cKO HSCs; CFU: colony-forming unit; Mk(or)E and G(or)M: mature megakaryocyte, erythroid, granulocyte or macrophage colonies; GM/Mix: immature GM or GMMkE colonies. Results are expressed as percent of plated cells. g–i, Scavenging ROS levels in Atg12^cKO mice: (g) scheme for NAC in vivo treatment after pIC deletion of Atg12, (h) neutrophil counts (left) and lineage distribution (right) in PB, and (i) quantification of immature BM populations. Data are mean ± S.D., and are expressed relative to Cnt HSC (a, b), or young HSC (d, e) levels.*p ≤ 0.05, **p ≤ 0.01.