Fig. 3

BTApep-TAT inhibited DNA damage repair governed by BORIS in cancer cells. (A) Artificial single-strand break DNA was incubated with crude nuclear extracts from HeLa cells transfected with full-length BORIS or the AA 1–258 N-terminus of BORIS (BORIS-N_1-258). Crude nuclear extracts of empty vector-transfected cells or equal volumes of water were used as negative controls. The DNA repair activities were examined by qRT–PCR and are shown as the relative transcript amount of ligated target A templates. (B) SSBR activities were examined in BORIS-expressing crude nuclear extracts that were treated with 25 µM peptides. (C) BER activities were examined in BORIS-expressing crude nuclear extracts that were treated with 25 µM peptides. The boiled nuclear extracts from BORIS-expressing HeLa cells were used as another negative control. The DNA repair activities were examined by qRT-PCR and are shown as the relative transcript amount of ligated target B templates. (D) The Xho I-linearized plasmid was used as double-strand DNA break to evaluate NHEJ activity. The multimeric and dimeric forms of the plasmids were visualized on agarose gels, which are presented in the left panel. The right panel shows the statistical summary of the treatment results. (E) DNA damage repair reporters (GFP fluorescent-based DNA repair reporter system) were used to evaluate the effect of BORIS in cells. The percentage of cells that underwent DNA damage repair was compared between BORIS-RFP-transfected cells and cells without transfection. DNA damage repair, which includes alternative NHEJ, total NHEJ, and homology-directed repair (HDR), was analyzed by flow cytometry