HSCs from Vav-Atg7^−/− BM fail to reconstitute the hematopoietic system of lethally irradiated mice. (A) Relative Atg7 messenger RNA (mRNA) expression in murine long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and LMPPs was measured by real-time Q-PCR. Data are mean ± SEM (n = 3). (B) CFC assay performed on total BM cells from WT (Vav-iCre⁺; Atg7^Flox/WT or Vav-iCre⁻; Atg7^Flox/Flox) and Vav-Atg7^−/− (Vav-iCre⁺; Atg7^Flox/Flox) mice. The graph shows the mean number of CFC colonies ± SD counted on day 12 (n = 3). (C) The methylcellulose cultures shown in B were replated in methylcellulose 12 d after initial plating. The total number of colonies was counted after 10 d in culture. Mean values ± SD (n = 3) are shown. (D) Competitive repopulation assay. CD45.2⁺ BM cells from WT (left) or Vav-Atg7^−/− (right) 9-wk-old mice were mixed 1:1 with CD45.1⁺ WT competitor BM cells and transplanted into lethally irradiated CD45.1⁺ WT recipients (n = 6 per group). Representative dot plots illustrating the CD45.2⁺ population frequency of donor-derived cells in peripheral blood of the recipient mice 16 wk after transplantation are shown. (E) Mean percentage (±SEM) of donor-derived CD45.2⁺ cells in peripheral blood of CD45.1⁺ recipients (n = 6 per group) described in D analyzed at 4, 12, and 16 wk after transplantation. (F) Lethally irradiated recipients were transplanted with 2 × 10⁶ WT BM cells alone (n = 3), Vav-Atg7^−/− BM cells (1.8 × 10⁶ cells) in a 10:1 ratio with CD45.1⁺ competitor BM (0.2 × 10⁶ cells; n = 4), or 2 × 10⁶ Vav-Atg7^−/− BM cells alone (n = 4). The mean percentage (±SEM) of CD45.2⁺ cells (gated as shown in D) in peripheral blood of the recipient mice is shown. Analysis was performed 4, 8, and 12 wk after transplantation. § indicates that recipients of Vav-Atg7^−/− BM cells alone had to be culled 4 wk after transplantation because of poor health, and no further analysis could therefore be performed. (G) Kaplan-Meier survival curves of recipient mice of the in vivo reconstitution assays described in F. (H) Kaplan-Meier survival curves of lethally irradiated recipients of either 10⁴ WT or Vav-Atg7^−/− flow-sorted BM LSK cells. (I) Lethally irradiated recipients were transplanted with 2 × 10⁶ WT (n = 5) or Vav-Atg7^−/− (n = 6) FL cells. The mean percentage (±SEM) of CD45.2⁺ donor cells in peripheral blood of the recipient mice over time is shown. The percentages of CD45.2⁺ myeloid (CD11b⁺Gr1⁺), B (B220⁺CD19⁺), and T cells (CD4⁺ and CD8⁺) are indicated (populations gated as shown in Fig. S1 F). Four out of six of the Vav-Atg7^−/− FL recipients had to be culled 7 wk after transplant because of their poor state. However, for simplicity, their percent blood populations are shown combined with those of the remaining recipient mice, which were bled 8 wk after transplantation. Two-tailed Mann-Whitney tests were performed on the indicated datasets (ns, nonsignificant; **, P < 0.01). (J) Kaplan-Meier survival curves of the lethally irradiated FL transplant recipients from I.