Figure 6: SWNTs specifically delocalize TRF2, POT1 and PCBP1 from telomeres and induce telomeric 3′-overhang degradation.

(a) HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹) were double stained with the indicated antibodies. Representative confocal images showing merged TRF1 (green) with TRF2, POT1 or PCBP1 (red) staining in untreated and treated cells. Scale bar equals 5 μM. (b) Percentages of cells with more than four colocalizations per nucleus of TRF1/TRF2, TRF1/POT1 or TRF1/PCBP1. The mean of three independent experiments with comparable results was shown. Error bars indicated s.d. **P<0.005. (c) Average number of colocalizations 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 TRF1, TRF2, POT1 or PCBP1 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. (e) Expression of TRF1, TRF2, POT1 and PCBP1 in HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹). β-actin was used as loading control. (f) Hybridization protection assay (HPA) was performed on genomic DNA isolated from HeLa cells expressing TRF2^ΔBΔM or treated with either 2′-O-MeRNA or SWNT-COOH (50 μg ml⁻¹) to assess the length of G-overhang and total telomere length. ExoI nuclease digestion was used to assess integrity of the 3′-overhang. Luminescence intensity in arbitrary units (AU) was normalized against Alu probe. The mean of three independent experiments with comparable results was shown. Error bars indicated±s.d., **P<0.01, two-tailed Student's t-test.