Serial repopulating defects and cell cycle abnormalities in Fancc^−/− fetal liver HSPCs. (A) Annexin V flow cytometry analysis for AA4.1⁺ Sca-1⁺ cells, P = .65. (B) Quantitative reverse-transcription-PCR gene expression analysis of Puma (p53-upregulated modulator of apoptosis) and Noxa (Phorbol-12-myristate-13-acetate-induced protein-1), pro-apoptotic, p53 target genes in Fancc^−/− and WT FL sorted ASL cells (n = 3/genotype), P_Puma = 0.17, P_Noxa= 0.13. (C) ROS flow cytometry analysis for AA4.1⁺ Sca-1⁺ cells, shown as median fluorescence intensity, P = .17. (D) Cell-cycle analysis for c-Kit⁺ Sca-1⁺ cells. Fancc^−/− average 45% decrease in Ki-67⁻ (G₀) cells, 34% increase in G₁, and 3% increase in S-G₂-M cells. Results normalized to WT littermates. (E) Quantitative reverse-transcription-PCR gene expression analysis of transcripts from sorted ASL or LSK cells from Fancc^−/− and WT FL. Bmi1 (BMI1 polycomb ring finger oncogene), Cdc42 (small GTPase cell division control protein42), p57 (cyclin-dependent kinase inhibitor 1C), EGR1 (early growth response 1), Gata3 (GATA binding protein 3), Hif-1α (hypoxia inducible factor-1α), and Elf4 (E74-like factor 4), N-cadh (N-cadherin). (F) Serial transplantation scheme: 2 × 10⁶ unfractionated CD45.2 FL cells were transplanted into conditioned CD45.1 recipients. Five months later, 2 × 10⁶ whole BM cells from 1° recipients were transplanted into CD45.1 hosts. (G) Percent ASL cells in WT and Fancc^−/− FL, P = .36. (H) Donor chimerism (% CD45.2⁺) of peripheral blood in 1° transplant (n_{WT FL donors} = 4, n_Fancc^−/−_{FL donors} = 3; n_{WT recipients} = 7, n_Fancc^−/−_recipients = 5), P = .13 for 1 month, .12 for 2 months, .10 for 3 months, and .14 for 5 months. (I) CD45.2⁺ chimerism (% FL donor-derived) for 2° transplantation. Two 1° recipients served as donors from each cohort (n_{WT recipients} = 10, n_Fancc^−/−_recipients = 10), P = .0002. Error bars represent standard error of the mean. BM, bone marrow; FL, fetal liver; WT, wild type.