Figure 5.

p65 is required to maintain cellular senescence in HFs. (A) HFs were sequentially infected with retroviruses expressing human TERT and pBabepuro (HF-TERT/V) or pBabe-IκBα-SR (HF-TERT/SR) in a total of eight independent lines (supplementary Fig S8D online). Cells were treated with 400 μM H₂O₂ for 10 days to induce cellular senescence and subsequently immortalized by using intermittent H₂O₂ treatment. Cell numbers were determined on a weekly basis for 50 weeks. (B) Karyotyping results of immortalized HF from line HF-TERT/SR1-1. Chromosomes 1 and 9 are shown to indicate the unbalanced translocation event, whereas chromosome 8 is shown as a reference. Arrowheads indicate break points. (C) Western blot of γ-H2AX in proliferating (P35) HF-TERT/V and HF-TERT/SR cells following IR similar to that described in Fig 3A. (D) Expression levels of γ-H2AX at indicated time points in (C) were quantified by NIH image software from three independent experiments. HF, human fibroblast; IR, γ-irradiation; NIH, National Institutes of Health; SR, super repressor; TERT, telomerase reverse transcriptase.