Expression of IRF-1, ISGF3γ, and p21^WAF1 is reduced in FA-C MEFs, B lymphoblasts, and FA patient LDBMCs.

(A) Immunoblot analysis of the FancC mutant MEF cell line (MEF61) shows increased expression levels of IRF-1, ISGF3g, and p21^WAF1 compared with the WT MEF cell line (MEF1.1) (lane 2 vs lane 1). The FANCC mutant B-lymphoblast line HSC536N, the HSC536N line transduced with a vector only (HSC536N/neo), and PD149 (lanes 4, 6, and 7, respectively) show increased IRF-1, ISGF3γ, and p21^WAF1 expression compared with the normal cell line JY (lane 3) and with cells corrected for the defect by retroviral transduction of the FANCC cDNA (HSC536N/FANCC) (lane 5) or PD149/FANCC (lane 8). However, FA-C cells express normal levels of IRF-2, p53, and p27^KIP1. To demonstrate equivalent loading of the ISGF3γ blots, β-tubulin is used. (B) Real-time reverse transcriptase PCR demonstrates up-regulation of MxA in HSC536N and HSC536N/neo compared with the corrected cells HSC536N/FANCC. This is a representative experiment. Error bars are based on duplicate samples. (C) The FA-C cell line HSC72 expresses equivalent levels of IRF-1 and ISGF3γ compared with cells corrected for the defect. The cells do express slightly more p21^WAF1, but equivalent p53. To demonstrate equivalent loading of the p21 blot, β-tubulin is used. (D) LDBMCs derived from 2 FA patients show up-regulation of IRF-1, ISGF3γ, and p21^WAF1 compared with cells derived from normal individual (lanes 1 and 3 vs lane 2).