Expression of FANCJ-A349P fails to rescue mitomycin C sensitivity of FA-J cells and exerts a dominant-negative effect on mitomycin C resistance of wild-type cells. (A) Western blot analysis of human FA-J (EUFA30/hTert/SV40) fibroblasts stably transfected with plasmids encoding GFP, GFP-tagged FANCJ-WT, or GFP-tagged FANCJ-A349P. Protein was detected with antibody against FANCJ (Sigma-Aldrich), GFP (Clontech) or actin (loading control, 10% loaded). (B) MMC-induced GFP-tagged FANCJ foci formation. GFP fluorescence detection was performed on FA-J cells transfected with plasmids encoding GFP, GFP-tagged FANCJ-WT, or GFP-tagged FANCJ-A349P. FA-J cells were treated with or without 100nM MMC for 2 hours. (C) Quantitative analyses of GFP-tagged FANCJ foci shown in panel B. (D) MMC sensitivity of stably transfected human FA-J fibroblasts with indicated genotypes was evaluated by cell survival assay. (E) γ-H2AX immunofluorescence staining of FA-J cells transfected with plasmids encoding GFP, GFP-tagged FANCJ-WT, or GFP-tagged FANCJ-A349P. Stably transfected FA-J cells were treated with or without 100nM MMC for 2 hours, washed with phosphate-buffered saline (PBS), allowed to recover for 6 hours, and analyzed by immunofluorescence detection as described in supplemental Methods. (F) Quantitative analyses of γ-H2AX foci shown in panel E. Data represent mean of > 100 cells; SD indicated by error bars. (G) Western blot analysis of human wild-type (VH10/hTert) fibroblasts transfected with plasmids encoding GFP, GFP-tagged FANCJ-WT, or GFP-FANCJ-A349P. Protein was detected with antibody against FANCJ (resolved on 6% SDS-PAGE), GFP, or actin (resolved on 4%-12% gradient SDS-PAGE). (H) MMC sensitivity of wild-type fibroblasts with indicated genotypes was evaluated by survival assay. (I) γ-H2AX immunofluorescence staining of wild-type fibroblasts transfected with plasmids encoding GFP, GFP-tagged FANCJ-WT, or GFP-tagged FANCJ-A349P. Wild-type cells were either untreated or exposed to 100nM MMC for 2 hours, washed with PBS, allowed to recover for 6 hours, and analyzed by immunofluorescence detection. (J) Quantitative analyses of γ-H2AX foci in the corresponding transfected wild-type cell lines as shown in panel I.