Senescent MEFs resemble oxidant-treated human fibroblasts. (a) Replicative lifespan, senescence (arrow) and spontaneous immortalization (resumed increase in cell number) is shown for the average of three independent C57Bl/6 MEF cultures (±s.d. indicated by error bars). (b) The percentage of labelled nuclei for a C57Bl/6 MEF culture were calculated at each passage for 55 days. Three independent cultures gave similar results. (c) Either early passage (PD2) or senescent (PD9) C57Bl/6 MEFs were analysed by western blotting for c-Fos and α-tubulin (control). Proteins (30 μg) from proliferating (lane 1), serum-deprived (lanes 2, 4) or serum-stimulated (lanes 3, 5) were analysed. (d) 82-6 human fibroblasts were treated with 400 μM hydrogen peroxide for 2 h. After 7 days, control and treated cells were maintained in 10% FCS (lanes 1 and 4), serum-deprived (lanes 2 and 5) or serum-deprived and stimulated (lanes 3 and 6). Proteins were analysed by western blotting for c-Fos and α-tubulin. c-Fos induction was detected 7 and 14 days after treatment with 200, 400 or 550 μM hydrogen peroxide. (e) Human fibroblasts were induced to senesce by infection with pLXSN-p14^ARF (ref. ) or treatment with 20 μg ml⁻¹ bleomycin for 2 h (ref.). After 7 days, control (untreated), infected (ARF) and treated (Bleo) cells were serum-deprived (lanes 1, 3 and 5) or serum-stimulated (lanes 2, 4 and 6) and analysed by western blotting for c-Fos and α-tubulin.

Low oxygen abolishes replicative senescence of MEFs. C57Bl/6 MEFs were used for all experiments. (a) MEFs were cultured in 20% or 3% oxygen, as indicated, and cell number was determined at each passage. The average and standard deviations of three independent cultures are shown. (b) MEFs cultured in 3% oxygen (black) were shifted at PD8, PD13 or PD19 to 20% oxygen (grey), or maintained in 3% oxygen. Cell number was determined at the indicated times. The average of two cultures is shown. (c) MEFs cultured in 3% or 20% oxygen were assayed for levels of p16, p19^ARF and α-tubulin (control) by western blotting. 3% oxygen cultures were analysed at early (PD2) and late (PD14 and PD24) passage. A senescent 20% oxygen culture (PD9) is shown for comparison. (d) Six 3% oxygen MEF cultures at the indicated population doublings were analysed for levels of p53 and α-tubulin by western blotting before (−) or 1 h after X irradiation (+; 4.5 Gy). (e) An early passage (PD5) and two late-passage (PD36 and PD38) 3% oxygen MEF cultures were analysed for levels of p21 and α-tubulin before (−), 6 h and 14 h after X irradiation (4.5 Gy). (f) Six 3% oxygen MEF cultures at the indicated population doublings and two 20% oxygen immortal cultures were infected with control (black bars) or p19^ARF-expressing (grey bars) retroviruses. After 48 h, ³H-thymidine was added for 1 h and the S-phase fraction determined (percentage labelled nuclei). (g) An early passage (PD5) and two late-passage (PD36 and PD38) 3% oxygen MEF cultures were infected with insertless vector (V) or Ha-Ras^V12-expressing (R) retroviruses and assayed for p19^ARF, p53, Ras and α-tubulin by western blotting. (h) Exponentially growing (+) and serum-deprived (−) early passage (PD5) and late-passage (PD36 and PD38) 3% oxygen MEFs were assayed for levels of Rb by western blotting. Equal loading was confirmed by Ponceau S staining.

Growth of repair-deficient MEFs in 3% and 20% oxygen. (a) The replicative lifespans of three independent Balb/c MEF cultures maintained in 3% oxygen and two cultures maintained in 20% oxygen are shown. Error bars indicate the standard deviations. (b) Replicative lifespans and standard deviations of five Ku80^−/− (grey) and three wild-type littermate (black) MEF cultures in 3% (circles) and 20% (squares) oxygen were determined. (c) The replicative lifespans and standard deviations of two Xpa^−/− (grey) and two wild-type littermate (black) MEFs cultures grown in 3% (circles) and 20% (squares) oxygen are shown.

MEFs accumulate high levels of oxidative DNA damage in 20% oxygen. (a) The Fpg-comet assay was performed on early passage C57Bl/6 MEFs cultured in 3% or 20% oxygen, immortal MEFs cultures derived and grown in 20% oxygen and mid-lifespan (PD 35) WI-38 human fibroblasts cultured for more than 10 population doublings in 20% oxygen. The normalized average tail length is plotted. Each value represents the average of four independent experiments and at least 50 cells per determination. (b) Comet assay controls. WI38 and MEFs grown at the indicated oxygen concentrations and at the indicated population doublings were analysed for comet tail lengths before treatment with fpg. As a positive control, the normalized tail length of MEFs treated with hydrogen peroxide (100 μM) is also shown. (c) C57Bl/6 MEFs were derived and grown in 3% or 20% oxygen for up to 25 passages. The average percentage of metaphases from 4–5 independent cultures containing obvious chromosomal breaks or fragments is plotted. Error bars show s.e.m.