126 Aging, protein solubility and lifespan in C. elegans, P. Reis-Rodrigues et al. Western blots Extraction of insoluble protein was preformed as described earlier. Total protein levels were measured by BCA assay (Thermo Fisher Scientiﬁc), and loading amounts were normalized. SDS-PAGE was performed using 4–12% Bis–Tris gel NuPAGE system (Invitrogen, Carlsbad, CA, USA). DAF- 21 antibody was obtained from Enzo Life Sciences (Plymouth Meeting, PA, USA) and Aconitase-2 antibody from Abcam (Cambridge, MA, USA). RNA interference RNA interference was undertaken as previously described (Olsen et al., 2006). Brieﬂy, worms grown at 20 C were used to lay eggs. Synchro- nized L4 larvae were moved to RNAi plates (NGM containing 100 lg mL)1 of ampicillin, 20 lg mL)1 tetracycline, and 1 mM IPTG) spotted with bacteria expressing double stranded RNAi. hsf-1 RNAi was from the Ahringer library and conﬁrmed by sequencing. Adult lifespan assays Wild-type worms were grown in mass cultures in enriched peptone plates with NA22 as food source (Strange et al., 2007). Eggs were synchronized by bleaching gravid adults (Fabian & Johnson, 1994) and arresting L1 larvae in S- Basal overnight. Worms were then grown in regular NGM plates with OP50 as food source and transferred to RNAi plates (NGM containing 100 lg mL)1 of ampicillin, 20 lg mL)1 tetracycline, and 1 mM IPTG) at late L4 stage. Most RNAi clones were from Open Biosystems library and when not available the Ahringer library clones were used. All RNAi treatments that extended lifespan were repeated at least three times. Data analysis was carried out using Graph- Pad Prism 4 (GraphPad Software Inc., San Diego, CA, USA). Gene ontology analysis GO analysis was carried out using NIH DAVID Bioinformatic Database (http://david.abcc.ncifcrf.gov/). The 203 proteins that were identiﬁed as being enriched in aged C. elegans were queried against the total protein C. elegans proteome. Subsequent lists of signiﬁcantly enriched biological process and cellular component GO categories were ﬁltered to exclude reported GO categories with a false discovery rate >0.05 and parsed of redundant categories. Acknowledgments We would like to acknowledge the expert technical assistance of the Chemistry Core of the Interdisciplinary Research Center on Gero- science and the Buck Institute for Research on Aging. We would also like to thank Matt Gill and Di Chen for valuable discussions and Michael Cusack for bioinformatics support. This work was supported by NIH Grants RL1 NS084432 (REH); 1R01AG029631-01A1 (GJL), R01 LM009722 (SDM), and the Larry Hillblom Foundation (REH and GLJ). Mass spectrometry was supported by an NCRR shared instru- mentation grant S10 RR024615 (BWG) and the Geroscience Mass Spectrometry and Imaging Core Grant PL1 AG032118 (BWG). SA was supported by the U19AGO231222 from the Longevity Consor- tium. TWP was supported by NIH Training Grant T32 AG000266. Author contributions PR-R, BWG, GJL, and REH designed the experiments. PR-R, GC, SA, EAG, and MV performed the experiments. PR-R, GC, TWP, USE, SDM, BWG, GJL, and REH analyzed data. PR-R, TWP, BWG, GJL, and REH wrote the paper. References Alavez S, Vantipalli MC, Zucker DJ, Klang IM, Lithgow GJ (2011) Amyloid-bind- ing compounds maintain protein homeostasis during ageing and extend life- span. Nature 472, 226–229. Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for dis- ease intervention. Science 319, 916–919. Ben-Zvi A, Miller EA, Morimoto RI (2009) Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc. Natl. Acad. Sci. U S A 106, 14914–14919. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A (2006) Opposing activities protect against age-onset proteotoxicity. Science 313, 1604–1610. David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C (2010) Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biol. 8, e1000450. Drake J, Link CD, Butterﬁeld DA (2003) Oxidative stress precedes ﬁbrillar deposi- tion of Alzheimer’s disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiol. Aging 24, 415–420. Fabian T, Johnson TE (1994) Production of age-synchronous mass cultures of Caenorhabditis elegans. J. Gerontol. Biol. Sci. 49, B145–B156. Garigan D, Hsu AL, Fraser AG, Kamath RS, Ahringer J, Kenyon C (2002) Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics 161, 1101–1112. Henderson ST, Johnson TE (2001) daf-16 integrates developmental and environ- mental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr. Biol. 11, 1975–1980. Hsu AL, Murphy CT, Kenyon C (2003) Regulation of aging and age-related dis- ease by DAF-16 and heat-shock factor. Science 300, 1142–1145. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366, 461–464. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G (1997) daf-2, an insulin receptor- like gene that regulates longevity and diapause in Caenorhabditis elegans. Sci- ence 277, 942–946. Lin K, Dorman JB, Rodan A, Kenyon C (1997) daf-16: an HNF-3 ⁄ forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Link CD (1995) Expression of human beta-amyloid peptide in transgenic Caenor- habditis elegans. Proc. Natl Acad. Sci. USA 92, 9368–9372. Link CD, Fonte V, Hiester B, Yerg J, Ferguson J, Csontos S, Silverman MA, Stein GH (2006) Conversion of green ﬂuorescent protein into a toxic, aggregation- prone protein by C-terminal addition of a short peptide. J. Biol. Chem. 281, 1808–1816. McColl G, Roberts BR, Gunn AP, Perez KA, Tew DJ, Masters CL, Barnham KJ, Cherny RA, Bush AI (2009) The Caenorhabditis elegans A beta 1-42 model of Alzheimer disease predominantly expresses A beta 3-42. J. Biol. Chem. 284, 22697–22702. Morimoto RI (2008) Proteotoxic stress and inducible chaperone networks in neu- rodegenerative disease and aging. Genes Dev. 22, 1427–1438. Morimoto RI, Cuervo AM (2009) Protein homeostasis and aging: taking care of proteins from the cradle to the grave. J. Gerontol. A Biol. Sci. Med. Sci. 64, 167–170. Morley JF, Morimoto RI (2004) Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones. Mol. Biol. Cell 15, 657–664. Morley JF, Brignull HR, Weyers JJ, Morimoto RI (2002) The threshold for polyglu- tamine-expansion protein aggregation and cellular toxicity is dynamic and inﬂuenced by aging in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 99, 10417–10422. Morrow G, Samson M, Michaud S, Tanguay RM (2004) Overexpression of the small mitochondrial Hsp22 extends Drosophila life span and increases resis- tance to oxidative stress. FASEB J. 18, 598–599. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G (1997) The Fork head transcription factor DAF-16 transduces insulin-like meta- bolic and longevity signals in C. elegans. Nature 389, 994–999. Olsen A, Vantipalli MC, Lithgow GJ (2006) Checkpoint proteins control survival of the postmitotic cells in Caenorhabditis elegans. Science 312, 1381–1385. Published 2011. This article is a U.S. Government work and is in the public domain in the USA. Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland