Figure 2
Figure 2. Human FANCC fails to complement DNA repair defects in Fancc-deficient cells but is sufficient for suppression of cytokine production. (A) Survivals of transformed Fancc-deficient tongue epithelial cells (6640SV/pLXSH) transduced with hFANCC (6640SV/hFANCC) or mFancc (6640SV/mFancc) and treated with MMC for 5 days (mean ± SD from one similar experiment of 3 performed in triplicate). 6640SV/hFANCC cells were significantly more sensitive to MMC than 6640SV/mFancc cells (P < .001; ANOVA). 6640SV/hFANCC cells were significantly more resistant to MMC than 6640SV cells (P < .001; ANOVA). (B) Colony assays of Fancc−/− hematopoietic progenitor cells (HPC-M) transduced with hFANCC (HPC-M/hFANCC) or mFancc (HPC-M/mFancc) and treated with MMC for 7 days (mean ± SD from one similar experiment of 2 performed in triplicate). HPC-M/hFANCC cells were 3-fold more sensitive to MMC than HPC-M/mFancc cells (P < .001; ANOVA), 6-fold more sensitive than wild-type cells (HPC-W; P < .001; ANOVA), and 3-fold more resistant to MMC than untransduced cells (HPC-M; P < .001; ANOVA). Transduction efficiencies of hFANCC/mFancc were measured by polymerase chain reaction with primers directed against green fluorescence protein and were similar (74% vs 79% for hFANCC and mFancc, respectively). (C) Immunoblots of fibroblast (top) or tongue epithelial (bottom) cell lines treated ± 150nM MMC for 24 hours. Lysates were blotted with either anti-FANCC antibody (top) or anti-Fancd2 antibody (bottom). (Top) Lanes 5 to 7 (NFF6, 11Lu, and NFF6/U195) represent normal human fibroblasts. The expression of hFANCC in transduced-murine fibroblasts (MEF61/hFANCC; lane 3) was more than 8-fold higher (by comparative densitometric analysis of lanes 3 and 7) than the level of expression required for a normal MMC response. WT indicates wild-type. (Bottom) Fancd2-L indicates mono-ubiquitinated Fancd2; and Fancd2-S, nonubiquitinated Fancd2. Fancd2−/− cells were isolated from Fancd2-deficient mice. (D) Primary (GFAC5 and 2) and transformed MEFs (MEF61) were treated with indicated concentrations of LPS for 24 hours. IL-6 secretion was measured in the supernatants by ELISA (mean ± SD from one similar experiment of 3 performed in triplicate). Fancc-deficient MEFs produced significantly higher levels of IL-6 than wild-type or transduced cells (P < .001 for both cell types).

Human FANCC fails to complement DNA repair defects in Fancc-deficient cells but is sufficient for suppression of cytokine production. (A) Survivals of transformed Fancc-deficient tongue epithelial cells (6640SV/pLXSH) transduced with hFANCC (6640SV/hFANCC) or mFancc (6640SV/mFancc) and treated with MMC for 5 days (mean ± SD from one similar experiment of 3 performed in triplicate). 6640SV/hFANCC cells were significantly more sensitive to MMC than 6640SV/mFancc cells (P < .001; ANOVA). 6640SV/hFANCC cells were significantly more resistant to MMC than 6640SV cells (P < .001; ANOVA). (B) Colony assays of Fancc−/− hematopoietic progenitor cells (HPC-M) transduced with hFANCC (HPC-M/hFANCC) or mFancc (HPC-M/mFancc) and treated with MMC for 7 days (mean ± SD from one similar experiment of 2 performed in triplicate). HPC-M/hFANCC cells were 3-fold more sensitive to MMC than HPC-M/mFancc cells (P < .001; ANOVA), 6-fold more sensitive than wild-type cells (HPC-W; P < .001; ANOVA), and 3-fold more resistant to MMC than untransduced cells (HPC-M; P < .001; ANOVA). Transduction efficiencies of hFANCC/mFancc were measured by polymerase chain reaction with primers directed against green fluorescence protein and were similar (74% vs 79% for hFANCC and mFancc, respectively). (C) Immunoblots of fibroblast (top) or tongue epithelial (bottom) cell lines treated ± 150nM MMC for 24 hours. Lysates were blotted with either anti-FANCC antibody (top) or anti-Fancd2 antibody (bottom). (Top) Lanes 5 to 7 (NFF6, 11Lu, and NFF6/U195) represent normal human fibroblasts. The expression of hFANCC in transduced-murine fibroblasts (MEF61/hFANCC; lane 3) was more than 8-fold higher (by comparative densitometric analysis of lanes 3 and 7) than the level of expression required for a normal MMC response. WT indicates wild-type. (Bottom) Fancd2-L indicates mono-ubiquitinated Fancd2; and Fancd2-S, nonubiquitinated Fancd2. Fancd2−/− cells were isolated from Fancd2-deficient mice. (D) Primary (GFAC5 and 2) and transformed MEFs (MEF61) were treated with indicated concentrations of LPS for 24 hours. IL-6 secretion was measured in the supernatants by ELISA (mean ± SD from one similar experiment of 3 performed in triplicate). Fancc-deficient MEFs produced significantly higher levels of IL-6 than wild-type or transduced cells (P < .001 for both cell types).

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