Research Paper Volume 2, Issue 9 pp 582—596

ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells


Figure 6. XRCC4 knockdown and expression of a XRRC4 decoy partially reduces NHEJ in hESCs. (A) XRCC4 knockdown and NHEJ in hESCs. Western blot analysis of extracts with XRCC4 antibody was carried out 48 and 72 h after transfection of BG01V/NHEJ-red cells with non-targeted control siRNAs or siRNAs targeting XRCC4. The fold change in XRCC4 levels was calculated after normalization to GAPDH which served as a loading control. (B) BG01V/NHEJ-red cells were infected with Ad-I-SceI at 30 MOI, 48 h after knockdown. Cells were collected at 24 h post-infection for genomic DNA qPCR to determine repair. (C) XRCC4 decoy reduces NHEJ in hESCs. Immunocytochemistry (top panel) and western blot (bottom panel) of BG01V/NHEJ-red cells 48 h after infection with the Ad-Flag-XRCC4115-293 virus described previously [31], or an EGFP expressing adenovirus. (D) BG01V/NHEJ-red cells were infected with either adenovirus for 48 h and then infected with Ad-SceI and harvested 24 h later. (Columns) Relative NHEJ levels were determined by qPCR and normalized to β-actin levels (Error bars) SEM of three samples. Fold (x) and statistical significance indicate changes in the relative repair levels as compared to those in the I-SceI-expressing cells treated with non-targeting control siRNA.