Research Paper Volume 2, Issue 9 pp 582—596

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


Figure 5. DNA-PKcs knockdown partially reduces NHEJ in hESCs. (A) Western blot showing DNA-PKcs expression 48 and 72 h after transfection of BG01V cells with GFP control siRNAs or siRNAs targeting DNA-PKcs or ATR [5]. The fold change in DNA-PKcs was calculated after normalization to ATR which served as a loading control together with a non-specific (N.S.) band. (B) Western blot showing HA-SceI levels in BG01V cells 48 h after infection which occurred 48 h after knockdown. (C) BG01V/NHEJ-red cells were infected with Ad-I-SceI at 30 MOI, 48 h after knockdown. DsRed events were determined by FACS 48 h after infection. (Columns) % DsRed+ cells with 10,000 events collected; (Error bars) SEM for data sets n = 3. (D) BG01V/NHEJ-red cells were infected with Ad-I-SceI at an MOI of 30 48 h after knockdown. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were determined by genomic DNA qPCR and normalized to β-actin levels; (Error bars) SEM for three samples. Fold (x) and statistical significance indicates changes in the relative repair levels compared to the siGFP sample.