Figure 4: DNA-damage response triggered by SWNT-COOH occurred at telomeres.

(a) HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹) for 2 weeks were fixed and processed for immunofluorescence using antibodies against γ-H₂AX (red)/TRF1 (green) or 53BP1 (green)/TRF1 (red), respectively. Representative confocal images were shown. Scale bar equals 5 μM. (b) TIF index, defined as foci of DNA-damage response factors that coincided with TRF1, was calculated as the percentage of TIF-positive cells in HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹). Cells with four or more γ-H2AX/TRF1or 53BP1/TRF1 foci were scored as TIF-positive. The mean of three independent experiments was reported. Error bars indicate s.d. **P<0.001, two-tailed Student's t-test. (c) Average number of TIFs per nucleus in HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹). The mean of three independent experiments with comparable results was shown. Error bars indicated±s.d. **P<0.005, two-tailed Student's t-test. (d) Binding of γ-H2AX and 53BP1 was examined by ChIP assay and detected by qRT–PCR amplification of the telomeric region in HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹). Data represented triplicate ChIP experiments, each with technical triplicates of qRT–PCR; **P<0.01 as compared with controls.