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Blood, Vol. 114, Issue 1, 174-180, July 2, 2009
Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M
Blood Singh et al.
114: 174
Supplemental materials for: Singh et al
Files in this Data Supplement:
- Figure S1 (JPG, 358 KB)
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(A) Ectopic expression of FANCM is not sufficient to restore FANCD2 monoubiquitination in EUFA867 cells. Lymphoblasts were transduced with retroviruses of either VSG or RD114 pseudotypes expressing FLAG-tagged FANCM. The transduced cells were either treated with 1mM HU for 16 h or left untreated and the total lysates were immunoblotted for FANCD2 and FANCM proteins. HSC93 (wild type) and EUFA867 lymphoblasts were used as positive and negative controls respectively. (B) Cell fusion between EUFA867 and FANCA deficient HSC72 lymphoblasts does not correct the MMC hypersensitive phenotype of EUFA867 cells. Lymphoblast were continuously exposed to different doses MMC and cell growth was compared to untreated cells by cell counting. Four independent cell fusions are depicted. HSC72 is the reference cell line for FA complementation group A and is able to complement the MMC hypersensitive phenotype of all FA cell lines, except those that have a defect in FANCA. HSC93 is included as wild type control lymphoblasts. (C) Cell fusion between EUFA867 and FANCC deficient HSC536 lymphoblasts does correct the MMC hypersensitive phenotype of EUFA867 cells. Two independent cell fusions are depicted. HSC536 is the reference cell line for FA complementation group C and is able to complement the MMC hypersensitive phenotype of all FA cell lines, expect those that have a defect in FANCC. HSC93 is included as wild type control lymphoblasts.
- Figure S2. Pedigree of the EUFA867 family and overview of mutations and clinical phenotype (JPG, 328 KB)
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Fanconi anemia patient EUFA867 (subject II:1) has two pathogenic mutations in FANCM and two pathogenic mutations in FANCA . The nonsense mutation S724X in exon 13 of FANCM is inherited from the mother, while the large genomic deletion leading to a frame shift by skipping of exon 15 sequence is not found in blood DNA from the father and probably arisen in the germ line of the father. The splice site mutation in FANCA (IVS7+5G>A) is inherited from the mother, the nonsense mutation (R853X) is inherited from the father. EUFA867 has one affected brother (subject II:3) who carries the two FANCA mutations and only one of the FANCM mutations. This patient was first diagnosed as a typical FA patient. The phenotype of this patient seems more severe than that of EUFA867.
- Figure S3. Stable expression of FANCA in EUFA867 lymphoblasts partially corrects the cellular phenotype (JPG, 203 KB)
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(A) Stable transfection of EUFA867 lymphoblasts with FANCA cDNA does not restore MMC hypersensitivity. EUFA867 cells were stably transfected with episomal vector pMEP4 encoding FANCA-flag. Lymphoblasts were continuously exposed to different doses MMC and cell growth was compared to untreated cells. Wild type (HSC93) and FANCA deficient (HSC72) lymphoblasts are included as controls. (B) Stable transfection of EUFA867 lymphoblasts with FANCA cDNA partially corrects FANCD2 monoubiquitination. Wild type lymphoblasts (HSC93), untransfected EUFA867 lymphoblasts (EUFA867) and FANCA transfected EUFA867 lymphoblasts (EUFA867+FANCA) were treated for 16 hours with 5 µM aphidicolin, 320 µM HU or 200 nM MMC. Whole cell extracts were investigated for FANCD2 monubiquitination and FANCA expression by western blotting.
- Figure S4. Camptothecin sensitivity in lymphoblastoid cell lines from different FA complementation groups (JPG, 808 KB)
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(A) Lymphoblasts with a defect in FANCA are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts. (B) Lymphoblasts with a defect in FANCB or FANCJ are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts, while lymphoblasts with a defect in BRCA2 are as sensitive as EUFA867 cells. (C) Lymphoblasts with a defect in FANCC and cell fusions between FANCC deficient cells and EUFA867 are as sensitive to growth inhibition as wild type lymphoblasts. (D) Lymphoblasts with a defect in FANCD2 are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts. (E) Lymphoblasts with a defect in FANCE are as sensitive to growth inhibition with different doses camptothecin as functionally corrected FA-E lymphoblasts (EUFA410+FANCE). Cell line EUFA410 was functionally corrected by stable transfection with vector pIRESneo encoding HA-tagged FANCE. (F) Lymphoblasts with a defect in FANCG are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts. (G) Lymphoblasts with a defect in FANCI are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts. (H) Lymphoblasts with a defect in FANCL are as sensitive to growth inhibition with different doses camptothecin as wild type lymphoblasts.
- Figure S5. Ectopic expression of FANCM is sufficient to restore camptothecin resistance in EUFA867 cells (JPG, 89.3 KB)
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Wild type, EUFA867 and EUFA867 cells transduced with FANCM were continuously exposed to different doses of camptothecin as indicated and viable cells were measured with the Cell Titre 96 Proliferation Assay.
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