Research Paper Volume 2, Issue 9 pp 582—596

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


Figure 4. Specific DNA-PKcs and ATM kinase inhibitors become more effective as hESCs differentiate. (A) DNA-PKi and ATMi are functioning in hESCs. hESCs were harvested 5, 10, and 15 min after exposure to 6 Gy with or without ATMi (10 μM) and DNA-PKi (2.5 μM) or both. Drugs were added 15 min prior to radiation. Fold change depicts phosphorylation of KAP1 (S824) and H2AX (S139) after normalization to CHK1 (and GAPDH) which served as loading controls. (B) BG01V/NHEJ-red (C) NP/NHEJ-red and (D) astrocyte/NHEJ-red cells were infected with Ad-SceI and then treated with either ATMi at 10 μM or DNA-PKi at 2.5 μM 1 h after infection. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were normalized to β-actin; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. Differences in the scale of the separate cell populations (B-D) are due to variation in the uninfected sample PCR amplification from 3 separate experiments. Statistical significance of differences in NHEJ with respect to cells expressing I SceI with no inhibitor, are indicated.