Fig. 3. Effect of uranyl nitrate on DNA double-strand break repair. (A) Diagram of the homologous recombination (HR) reporter assay. The DR-GFP plasmid carries a non-functional mutant GFP (SceGFP) and an internal truncate of GFP (iGFP). When transfected with an I-SceI along with an RFP-expressing plasmid to control transfection efficiencies, I-SceI excised SceGFP to induce DSBs.30 Only by HR repair with iGFP as the homologous template could restore the damaged SceGFP to a functional GFP. GFP⁺ cells were HR-repair cells. (B) Diagram of the NHEJ reporter assay. EJ5-GFP contains a promoter that is separated from a GFP-coding cassette by a puro gene that is flanked by two HindIII sites. Before transfection, the EJ5-GFP plasmid was linearized by HindIII enzyme digestion. Only by NHEJ repair could join the promoter and the GFP sequence, and GFP⁺ cells were NHEJ-repaired cells.31 (C) A representative of the flow cytometry measurements for HR pathway activity. (D) Quantification of the HR activity. Relative HR efficiency was measured by the percentage of GFP⁺ cells among RFP⁺ cells. (E) A representative of the flow cytometry measurements for NHEJ pathway activity. The pCherry plasmids also expressed the red fluorescence protein (RFP). (F) Quantification of the NHEJ assay. Relative NHEJ efficiency was measured by the percentage of GFP⁺ cells among pCherry⁺ cells.*P < 0.05, **p < 0.01.