Mechanism of increased susceptibility of Fanca^−/− HSPCs to KIT-mediated apoptosis and cell death. (A-C) Whole BM, LSK (% of BM), and LSK-SLAM cells (% of LSK) from 2 femurs (WT, n = 16: Fanca^−/−, n = 18). (D) Percentage cycling (Ki67⁺) HSPCs (LK [lin-KIT⁺], LSK, and LSK-SLAM [SLAM] cells) from WT and Fanca^−/− BM (WT, n = 5; Fanca^−/−, n = 5). (E-F) Expression of surface KIT (F) and total KIT in LSK cells (WT, n = 8; Fanca^−/−, n = 7). (G) Relative SCF mRNA expression in BM stroma (WT, n = 6; Fanca^−/−, n = 4). (H) mRNAseq analysis heat map of differentially expressed apoptosis and survival genes in WT and Fanca^−/− LSK cells post ACK2 (WT, n = 2; Fanca^−/−, n = 2). (I) Viable cells from a starting cell number of 10 000 LSK cells, sorted by flow cytometry, and cultured in X-vivo 10 containing 100 ng/mL each of recombinant murine SCF and thrombopoietin, with or without 50 mg/mL of ACK2. Cell counts at 72 hours are shown (n = 3 experiments). (J-K) Degree of apoptosis and cell death following 16 hours of culture of WT and Fanca^−/− LSK cells in X-vivo 10 medium containing 50 ng/mL each of recombinant murine SCF and thrombopoietin with or without 20 μg/mL ACK2 (WT, n = 4; Fanca^−/−, n = 4). ****P < .0001; ***P < .001; **P < .01; *P < .05 (Mann-Whitney U test). MFI, mean fluorescence intensity; ns, nonsignificant.