Ectopic HOXB4 protects HSC/P from the inhibitory effects of TNF-α. (A) Survival of transduced progenitor cells with increasing concentrations of TNF-α. Murine LSK cells were transduced with the indicated retroviral vectors, and sorted transduced cells were then plated in methylcellulose supplemented with 1 ng/mL, 10 ng/mL, or no (NTX) TNF-α. The frequency of surviving day 7 colonies, expressed as a percentage of nontreated controls, is shown. Data represent the least squares mean estimates plus or minus SEM from mixed-effects model analysis of 3 or 4 independent experiments. □ represents Fancc^−/− + SF-IG; , Fancc^−/− + SF-FAC-IG; ■, Fancc^−/− + SF-HOXB4-IG; , WT + SF-IG. *Significant compared with Fancc^−/− + SF-IG group or as indicated by bars after multiple comparison adjustment (P < .003) by Bonferroni method. NS indicates not significant. (B,C) Immunophenotypic analysis of percentage HSC/P in culture after TNF-α treatment. LSK BM from C57B6 or Fancc^−/− mice were transduced with either SF-IV or SF-HOXB4-IV. At 36 hours after transduction, cells were treated with the indicated dose of TNF-α as described in “Analysis of G₂/M arrest and FAN CD2 mono-ubiquitination in human FA lymphoblast cell lines” and a further 24 hours later BM was stained with antibodies directed against lineage markers, c-Kit and Sca-1. The percentage of BM cells corresponding to either (B) lin⁻ c-Kit⁺ or (C) lin⁻, c-Kit⁺, Sca1⁺ is shown. Data represent the least squares mean estimates plus or minus SEM from mixed-effects model analysis of 5 or 6 independent experiments. □ represents Fancc^−/− + SF-IV; ■, Fancc^−/− + SF-HOXB4-IV; , WT + SF-IV; ▩, WT + SF-HOXB4-IV. *Significant compared with nontreated control after multiple comparison adjustment (P < .05) by Tukey method.