Fig. 5.
Activation of FOXO3A inErcc1-/-mammalian cells mimics worms. (A) Representative image of crystal violet staining of cultures of passage 3 WT and Ercc1-/- primary MEFs 24 h after treatment with 0.5 mM paraquat. (B) Quantification of cell viability 24 h after treatment of passage 4 WT and Ercc1-/- MEFs with 0.5 mM paraquat (mean percent survival compared to untreated cells ± S.E.M.; n = 5 independent cultures/genotype). Student's two-tailed t-test * *p < 0.01. (C) Quantification of cell viability 24 h after treatment of passage 3 WT and Ercc1-/- MEFs with 1 µM rotenone (mean percent survival compared to untreated cells ± S.E.M. n = 5 replicas). Student's two-tailed t-test, * *p < 0.01. (D) Immunodetection of FOXO3A in nuclear (top) or cytosolic fractions (bottom) of primary MEFs at increasing passage number. -/- indicates Ercc1-/- cells. Lamin A/C is used as a loading control for nuclear proteins; β-tubulin is used for cytosolic. (E) Nonspecific oxidant levels in WT and Ercc1-/- MEFs measured with 2’,7’-dichlorofluorescein (H2-DCFDA) dye and flow cytometry at early (P3) and late (P5) passage of cells (n = 5 independent cell lines; data are represented as mean ± S.D.). Two-tailed Student's t-test * *p < 0.01, * **p < 0.05. (F) Detection of 2-hydroxyethidium (2-OH-E+) in WT and Ercc1-/- primary MEFs at passage 5 (mean ± S.D. of n = 8 independent cell pellets/genotype). Student's two tailed t-test ***p < 0.001.