1. Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair
by Luca Colnaghi, Mathew J. K. Jones, Xiomaris M. Cotto-Rios, Detlev Schindler, Helmut Hanenberg, and Tony T. Huang
Blood
Volume 117(7):
February 17, 2011
©2011 by American Society of Hematology

2. FANCI is mislocalized in FA-I F010191 lymphoblasts.
FANCI is mislocalized in FA-I F lymphoblasts. (A) Schematic representation of WT FANCI and mutant FANCI proteins expressed in F FA-I cells. In silico predicted leucine zipper, armadillo (ARM) repeat domain, EDGE motif and NLS, and lysine 523 (the monoubiquitination target of the FA core complex). (B) Time course of FANCI and FANCD2 monoubiquitination after 2mM HU treatment in normal or F LCLs evaluated by Western blot analysis with the indicated antibodies. Note the slightly faster migrating FANCI protein in the F LCLs resulting from the missing residues from the C-terminus. (C) Equal amounts of fractionated cytoplasmic (C) and nuclear (N) protein extracts from normal or FA-I lymphoblast lines were analyzed by Western blot with the indicated antibodies. Procedure for fractionation is described in “Subcellular fractionation.” (D) Nuclear accumulation of FANCI and FANCD2 proteins was quantified by determining the band intensity of underexposed Western blot film with ImageJ Version 1.43 software. Error bars represent SD from 3 independent experiments. The percentage of protein in the nucleus is compared with total from both cytoplasmic and nuclear fractions.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology

3. Exogenous expression of FANCI R1299X mutant localizes to cytoplasm in U2OS cells.
Exogenous expression of FANCI R1299X mutant localizes to cytoplasm in U2OS cells. (A) Table showing FANCI and c-Myc NLS amino acid sequence used in the study. NLS software ( was used to identify the putative C-terminal NLS sequence for FANCI. (B) Schematic diagram of FANCI WT, FANCI R1299X, and FANCI R1299X proteins with the fusion of NLS sequences from FANCI (FANCI-NLS2) or c-Myc (FANCI-NLS3) at the C-terminus and C-terminal NLS deleted (FANCI-ΔNLS). (C) U2OS were transfected with the indicated Flag-tagged FANCI constructs. Forty-eight hours after transfection, cells were fixed and stained with anti-Flag antibody. 4,6-diamidino-2-phenylindole is used for DNA staining in the nucleus. A total of 200 cells were counted and scored for predominantly nuclear (N), both nuclear and cytoplasmic (N-C), or cytoplasmic (C) staining and graphed as percentage of total cells. Representative images of each expression constructs are shown.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology

4. Addition of an NLS sequence to FANCI R1299X can rescue FANCD2 monoubiquitination and nuclear foci formation after DNA damage.
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.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology

5. Recovery of FANCD2 monoubiquitination in the FA-I F010191 fibroblasts.
Recovery of FANCD2 monoubiquitination in the FA-I F fibroblasts. (A) HeLa and F fibroblasts were either untreated or treated with HU (2mM for 18 hours). (B-C) F fibroblasts were transfected with empty vector or the indicated FANCI expression constructs. Forty-eight hours after transfection, cells were treated with 2mM HU for 24 hours and FANCD2 monoubiquitination was assessed by Western blot analysis and probed with the indicated antibodies. The L/S ratio was calculated as described in Figure 3A.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology

6. FANCI missing the EDGE motif fails to correct MMC sensitivity in the FA-I F010191 fibroblasts.
FANCI missing the EDGE motif fails to correct MMC sensitivity in the FA-I F fibroblasts. (A) F fibroblasts were transfected with empty vector or the indicated FANCI expression constructs. Forty-eight hours after transfection, cells were treated with 2mM HU for 24 hours and processed for immunostaining. FANCD2 and BRCA1 nuclear foci formation was analyzed by immunofluorescence as previously described. (B) FANCD2 nuclear foci after HU treatment were quantified. Error bars represent SD from 3 independent experiments. (C) FA-I F fibroblasts were transfected with either empty vector or the indicated FANCI expression constructs; and 48 hours after transfection, cells were treated with MMC at the indicated dose for 5 more days. Percentage cell survival is shown as an average of 3 independent experiments. Error bars represent the SD for the different experiments performed.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology

7. A deletion of the C-terminal NLS of FANCI fails to correct MMC sensitivity in the FA-I F fibroblasts.
A deletion of the C-terminal NLS of FANCI fails to correct MMC sensitivity in the FA-I F fibroblasts. (A,C) F fibroblasts were transfected with empty vector or the indicated FANCI expression constructs. Forty-eight hours after transfection, cells were treated with 2mM HU for 24 hours and FANCD2 monoubiquitination was assessed by Western blot analysis and probed with the indicated antibodies. The L/S ratio was calculated as described in Figure 3A. (B,D) FA-I F fibroblasts were transfected with either empty vector or the indicated FANCI expression constructs; and 48 hours after transfection, cells were treated with MMC at the indicated dose for 5 more days. Percentage cell survival is shown as an average of 3 independent experiments. Error bars represent the SD for the different experiments performed.
Luca Colnaghi et al. Blood 2011;117:
©2011 by American Society of Hematology