FANCE is required for function of the Fanconi anemia pathway.

(A) Complementation of MMC sensitivity of an FA-E lymphoblast cell line, EUFA130, with pMMP puro FANCE or pMMP puro HA-FANCE. The indicated retroviral supernatants were generated and used to transduce FA lymphoblast lines. Puromycin-resistant cells were selected, and MMC sensitivity was determined as described in “Materials and methods.” (B) Reintroduction of FANCE or HA-FANCE in FA-E lymphoblasts restores the FANCA/FANCC interaction. Whole-cell extracts were generated from the lymphoblast lines, EUFA130, EUFA130+FANCE, and EUFA130+HA-FANCE. Equal amounts of protein from each extract (2 mg) were used for immunoprecipitation with affinity-purified anti-FANCA antibody. Immune complexes were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-FANCA, anti-FANCC, or anti-FANCG antibody. (C) FANCE is required for the monoubiquitination of FANCD2. Whole-cell extracts were prepared from the indicated lymphoblast cell lines. Some cell lines were harvested 8 hours after treatment with IR (15 Gy) or 24 hours after continuous exposure to MMC (40 ng/mL), as indicated. Cellular proteins were immunoblotted with anti-FANCD2, anti-FANCA, anti-FANCG, anti-FANCC, anti-FANCF, or anti-HA antibody. FANCD2-S (short) is a nonubiquitinated isoform, and FANCD2-L (long) is a monoubiquitinated isoform of FANCD2. (D) FANCE is required for nuclear foci formation of FANCD2. The indicated lymphoblasts treated with MMC (40 ng/mL) for 24 hours were double-immunostained with anti-FANCD2 and anti-HA antibodies. In FA-E lymphoblasts (EUFA130), FANCD2 is expressed in a diffuse nuclear pattern. Correction of FA-E cells with either FANCE cDNA or HA-FANCE cDNA restores FANCD2 nuclear foci. Counterstains for the DNA-specific dye, DAPI, are shown in the middle panels. Expression of HA-FANCE was confirmed, as shown in the lower panels. Original magnification, × 600.