Addition of an NLS sequence to FANCI R1299X can rescue FANCD2 monoubiquitination and nuclear foci formation after DNA damage. (A) Diagram of the 21-bp FANCI oligonucleotide siRNA target sequence (top line). Bold letters indicate silent mutations introduced in the siRNA-resistant FANCI cDNA (bottom line). U2OS cells were transfected with control or FANCI-specific siRNA for 24 hours, followed by transfection of empty vector or the indicated FANCI expression constructs. Twenty-four hours after the second transfection, cells were treated with 2mM HU for 18 hours and FANCD2 monoubiquitinatation was assessed by Western blot analysis with the indicated antibodies. The L/S ratio between the monoubiquitinated (L) and unmodified form of FANCD2 (S) is shown and measured using ImageJ Version 1.43 software. (B) Replacement of endogenous FANCI in U2OS with empty vector or the indicated FANCI expression constructs were performed as described in panel A. Cells were then treated with 2mM HU for 18 hours and FANCD2 and BRCA1 foci formation was analyzed by indirect immunofluorescence as indicated. FANCD2 nuclear foci after HU treatment were quantified as percentage of cells with 5 or more FANCD2 foci. Error bars represent the SD from 3 independent experiments.