ScribbleΔ/Δ hematopoietic reconstitution develops a competitive advantage when serially transplanted by maintaining self-renewal divisions. (A) Schematic representing a serial competitive repopulation assay (CRA). Equal amounts of BM from CD45.2+ Vav1Cre Wt and Scribble null mice mixed with congenic CD45.1+ B6.SJLPtprcaPep3b/BoyJ competitor cells were transplanted at a 1:1 ratio into lethally irradiated B6.SJLPtprcaPep3b/BoyJ recipients. (B-D) PB chimera (CD45.2+ leukocytes) of (B) primary, (C) secondary, and (D) tertiary recipients. (E) Schematic representing a long-term BrdU incorporation assay in which animals freely imbibed water containing BrdU (1 mg/mL) for 10 days. Animals were euthanized after 80 days post-BrdU administration to quantify quiescence within hematopoietic stem and progenitor (HSC/P) populations determined by BrdU retention (BD Pharmingen intracellular staining kit: anti-BrdU, Alexa 488). (F) Absolute BrdU retaining (BrdU+) HSC assessed by FACS analysis of BM from mice as described in panel E. (G) Division of sorted and individually deposited HSC depicting the averages and standard deviations of the relative number (%) of wells containing the indicated number of cells after 20 hours in culture. Four independent experiments, n > 450 HSC. (H) Schematic of an in vitro paired daughter cell assay to assess fate decisions among individually sorted HSC. (I) Representative cytospin images of paired daughters. m, macrophage; n, neutrophil; e, erythrocyte; and M, megakaryocyte. (J) Absolute number of paired daughter cells analyzed for division modality, assessed as the presence or absence of full multilineage differentiation potential among individual paired daughter clones. n of paired daughter separations >200. Chi-squared analysis. (K) Cell death analysis using Annexin V and 7-AAD staining on Rosa26Cre;Scribblefl/fl HSC after 40 hours of culture with 4-OH tamoxifen. (L) Cell death analysis using Annexin V staining in Lin− Sca-1+ c-kit+ BM cells isolated from Vav1Cre;Wt or Vav1Cre;ScribbleΔ/Δ mice transduced with EMPTY (Ef1α-IRES-RFP) lentivirus or Scribble structure-function mutants as indicated. (M) Immunofluorescence depicting fate determinant allocation of Myc and the corresponding HSC division mode in Wt and ScribbleΔ/Δ paired daughter HSC (nocodazole, 10 nM for 24 hours). Low Myc expression in (i) paired daughter cells represents symmetric self-renewal, (ii) high and low Myc expression between the 2 daughters represents an asymmetric division, whereas (iii) high Myc expression in both cells is indicative of symmetric commitment. (N) Quantification of fate determinant allocation and division mode among Wt and ScribbleΔ/Δ paired daughter HSC. *P < .05, **P < .01, and ***P < .001.