Figure 7: Cell cycle arrest, apoptosis and senescence evoked by carboxylated SWNTs-induced telomere dysfunction.

(a) Cell cycle arrest induced by carboxylated SWNTs in K562 and HeLa cells. Two weeks after treatment with SWNTs, cells were collected and stained with propidium iodide (PI); DNA content was determined by flow cytometry. The percentages of cells undergoing apoptosis (sub-G1%) were expressed with respect to the total number of cells. This assay was performed in triplicate. *P<0.05, **P<0.01. (b) Apoptotic cell death induced by carboxylated SWNTs in K562 and HeLa cells. Three weeks after treatment with SWNTs, cells were collected and stained with PI and Annexin V–FITC, Annexin V-positive/PI-negative cells were measured by flow cytometry. This experiment was repeated three times. **P<0.001. (c) Expression of senescence-associated β-galactosidase (SA-β-gal) in K562 and HeLa cells after continuous treatment with carboxylated SWNTs for 3 weeks. This assay was performed in triplicate. The senescent cells were counted under an inverted microscope in five random fields. **P<0.001. (d) Upregulation of p16 and p21 proteins induced by carboxylated SWNTs. After K562 and HeLa cells were treated with SWNT-COOH (50 μg ml⁻¹), 2′-O-MeRNA or transfection with TRF2^ΔBΔM for 3 weeks, cells were lysed and separated on 10% SDS–PAGE, and probed with anti-p16^INK4a and anti-p21^WAF1 primary antibody, respectively. Immunoblotting for β-actin was also performed to verify equivalent protein loading. Each experiment has been repeated three times.