(A) TNF-α–mediated selection for resistant Fancc^–/– BM cells. Lin-Sca-1⁺ BM cells from WT and Fancc^–/– mice were cultured in complete medium containing IL-11, IL-6, Steel factor, and Flt-3 ligand and in the presence or absence of 10 ng/ml TNF-α. The total number of viable cells was counted by the trypan blue exclusion method at the times indicated. Result shown are representative of 5 separate experiments with similar results and expressed as mean of duplicates. (B) Colony growth assays demonstrated that while Fancc^–/– BM progenitor cells were hypersensitive to TNF-α, the TNF-α–resistant Fancc^–/– clonal progenitors were resistant in clonal assays. Data represent total number of colonies (mean ± SD) from 3 independent experiments. (C) Expression and activation of the TNF family signaling molecules in WT, freshly isolated Fancc^–/–, and outgrown Fancc^–/– BM progenitor cells. Cells were cultured in cytokine-supplemented medium in the absence or presence of 10 ng/ml TNF-α for 12 hours. RNA and protein extracts were prepared, and gene expression (right) and activation of caspase-3 and -8 were analyzed by RT-PCR and immunoblotting, respectively. (D) Spectral karyotype (SKY) analysis of the TNF-resistant cells demonstrated clonal cytogenetic abnormalities, including a clone with t(10;11) and another with t(4;9). The images shown were obtained from 1 culture. We conducted cytogenetic analysis on 3 different cultures, which gave some shared (e.g., the 10;11 translocations and monosomy 14) as well as different aberrations.