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. Author manuscript; available in PMC: 2011 Dec 7.
Published in final edited form as: Curr Biol. 2010 Nov 18;20(23):2131–2136. doi: 10.1016/j.cub.2010.10.057

Fig. 4. Increased ROS causes HIF-1 to promote longevity in respiration-defective mutants.

Fig. 4

(A) ROS levels, measured using a 2′,7′-dichlorofluorescein diacetate (DCF-DA) fluorescence assay, were significantly increased in clk-1(qm30) and isp-1(qm150) mutants (n=5). See also Fig. S4A for data using mev-1(kn1) mutant animals, which were previously shown to have increased ROS levels [40]. (B–C) A low dose (0.25 mM) (B) and high dose (4 mM) (C) of paraquat significantly increased the level of DCF-DA fluorescence. 4 mM paraquat was introduced from L4 to day 3 of adulthood because worms arrest as larvae if treated with 4 mM paraquat from hatching (n=3). Error bars represent s.e.m (*P<0.05, **P<0.01, two-tailed Student’s t-test). (D) Low doses of paraquat (0.125 mM, 0.25 mM, 0.5 mM and 1 mM) lengthened lifespan, whereas higher concentrations (4, 16, and 64 mM) shortened lifespan. Paraquat was introduced during adulthood. The lifespan measurements for 0.125 mM and 0.5 mM paraquat treatment were performed separately and therefore shown with different controls. See also Table S4. (E, F) Compared to untreated control Pnhr-57::GFP animals (E), animals treated with low levels of paraquat (0.25 mM) displayed increased GFP levels (F). Paraquat treatment further increased Pnhr-57::GFP levels in clk-1(qm30) and isp-1(qm150) mutant animals (Fig. S4J, K), suggesting that the induction was not saturated by either of the mutations or by the paraquat treatment. (G, H) The induction of Pnhr-57::GFP by paraquat treatment was significantly diminished in the hif-1(ia4) mutant. (I) Quantification of fluorescence in E to H (n >15). (J) The increased nhr-57 mRNA abundance caused by paraquat treatment, assayed using quantitative RT-PCR, was significantly decreased by hif-1(ia4) mutation. Data analysis was done from 10 independent quantitative RT-PCR experiments. [In two of our data sets, the levels of nhr-57 mRNA in paraquat-treated wild-type animals were increased by a very-large 681 and 755 fold compared to those in control animals. We excluded these two datasets from our analysis by using rejection analysis of QP-test for outliers (confidence level: 0.99) [41]]. (K) Mutations in hif-1 significantly decreased the longevity caused by paraquat treatment. Some long-lived mutants require the daf-16/FOXO transcription factor gene for their longevity, but respiration mutants do not [1, 4, 10]. We found that 0.25 mM paraquat treatment increased the lifespan of daf-16(mu86) null mutants (Fig. S4L). See Supplemental Table S4 for statistical analysis. Error bars represent s.e.m (*P<0.05, **P<0.01, two-tailed Student’s t-test). Previously, Rea et al. observed an increased trend in protein carbonylation levels at doses of respiratory-chain RNAi that increased lifespan, and also at higher RNAi doses, which did not extend lifespan [17]. On this basis, they concluded that ROS did not play a role in this longevity. One way to reconcile their findings with ours is to suggest that a sharp reduction in respiration prevents lifespan extension in spite of elevated ROS because it compromises the animals’ health.