American Society of Hematology

Figure 3

$Figure 3. Ectopic expression of both FANCA and FANCM can correct the FA phenotype of EUFA867 lymphoblasts. (A) Ectopic expression of FANCA and FANCM in EUFA867 restores the chromatin localization of monoubiquitinated FANCD2, which coincides with an enhanced FANCD2 monoubiquitination. EUFA867 cells stably expressing FANCA (EUFA867-FANCA) were generated with pMMP-FANCA. Subsequently, these cells were transduced with either MIEG3 bicistronic retroviral vector or MIEG3 encoding wild-type FANCM. EGFP-positive cells were treated with 240 nM MMC for 16 hours or left untreated and subcellular fractions were made: S100 cytoplasmic and nucleoplasmic proteins, S400 chromatin-bound proteins. (B) The ATPase activity and the C-terminus of FANCM are not required for efficient FANCD2 monoubiquitination. EUFA867 lymphoblasts stably expressing FANCA were transduced with either MIEG3 retroviral vector or MIEG3 encoding wild-type FANCM, an ATPase-dead FANCM mutant (K117R-FANCM) or a C-terminal FANCM deletion mutant (delC-FANCM). EGFP-positive cells were treated with either 2 mM HU (top panel) or 240 mM MMC (bottom panel) for 16 hours. Total lysates were immunoblotted for FANCD2. (C) FANCM, but not its ATPase activity or its C-terminus, is required for the assembly of FANCD2 foci. Cells were either left untreated or exposed to 450 nM MMC or 2 mM HU for 24 hours, and the percentage of cells with 5 or more FANCD2 foci was determined in at least 150 cells. The result shows the average of 3 independent experiments with standard deviations. (D) The ATPase activity, but not the C-terminus of FANCM, is required for MMC resistance. Viable cells were measured with the Cell Titer 96 Proliferation Assay. The data represent the percentage growth compared with untreated cells and show 1 representative result of 3 independent experiments with standard deviations. (E) The FANCM ATPase mutant does not rescue the melphalan-induced G2 arrest in EUFA867 lymphoblasts. (F) FANCM is required for chromatin targeting of the FA core complex proteins. Subcellular fractions of EUFA867 lymphoblast and stably transduced derivatives were immunoblotted for FANCM, FANCA, FANCG, FANCL, and FAAP24. H2A was used as a loading control for the chromatin fraction.$

Ectopic expression of both FANCA and FANCM can correct the FA phenotype of EUFA867 lymphoblasts. (A) Ectopic expression of FANCA and FANCM in EUFA867 restores the chromatin localization of monoubiquitinated FANCD2, which coincides with an enhanced FANCD2 monoubiquitination. EUFA867 cells stably expressing FANCA (EUFA867-FANCA) were generated with pMMP-FANCA. Subsequently, these cells were transduced with either MIEG3 bicistronic retroviral vector or MIEG3 encoding wild-type FANCM. EGFP-positive cells were treated with 240 nM MMC for 16 hours or left untreated and subcellular fractions were made: S100 cytoplasmic and nucleoplasmic proteins, S400 chromatin-bound proteins. (B) The ATPase activity and the C-terminus of FANCM are not required for efficient FANCD2 monoubiquitination. EUFA867 lymphoblasts stably expressing FANCA were transduced with either MIEG3 retroviral vector or MIEG3 encoding wild-type FANCM, an ATPase-dead FANCM mutant (K117R-FANCM) or a C-terminal FANCM deletion mutant (delC-FANCM). EGFP-positive cells were treated with either 2 mM HU (top panel) or 240 mM MMC (bottom panel) for 16 hours. Total lysates were immunoblotted for FANCD2. (C) FANCM, but not its ATPase activity or its C-terminus, is required for the assembly of FANCD2 foci. Cells were either left untreated or exposed to 450 nM MMC or 2 mM HU for 24 hours, and the percentage of cells with 5 or more FANCD2 foci was determined in at least 150 cells. The result shows the average of 3 independent experiments with standard deviations. (D) The ATPase activity, but not the C-terminus of FANCM, is required for MMC resistance. Viable cells were measured with the Cell Titer 96 Proliferation Assay. The data represent the percentage growth compared with untreated cells and show 1 representative result of 3 independent experiments with standard deviations. (E) The FANCM ATPase mutant does not rescue the melphalan-induced G2 arrest in EUFA867 lymphoblasts. (F) FANCM is required for chromatin targeting of the FA core complex proteins. Subcellular fractions of EUFA867 lymphoblast and stably transduced derivatives were immunoblotted for FANCM, FANCA, FANCG, FANCL, and FAAP24. H2A was used as a loading control for the chromatin fraction.

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