In vivo NHEJ analysis. (A) Schematic illustration of the NHEJ reporter locus. The ATG start codon, located upstream of the I-Sce I recognition sites, is not in-frame with the GFP coding sequence. The relative location of the CMV promoter (PCMV) is indicated. (B) Analysis of the effect of hMSH4 on NHEJ. Plasmids encoding I-Sce I and the full-length hMSH4 or hMSH4 aa1-183 and aa848-936 fragments were co-transfected into the NHEJ reporter cell line 293T/#8-1. Transfected cells were analyzed by FACS at 48 hrs post-transfection. Average NHEJ activities of three independent experiments were graphed. Error bars are standard deviations from the means. (C) Dose-dependent effect of hMSH4 on NHEJ. Increased amounts of hMSH4 expression construct were transfected into NHEJ reporter cells at 24 hrs prior to I-Sce I transfection. FACS analysis was performed at 48 hrs post-I-Sce I transfection. Error bars represent standard deviations from the means of triplicate experiments. Western blotting analysis was performed to validate the increased levels of hMSH4 expression in the reporter cells.