Supporting Information

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Fig. 5. Both mek-1(ks54) and unc-16(e109) show little effect on lifespan. Lifespan analysis of loss-of-function mutant of mek-1(ks54) and hypomorphic allele of unc-16(e109). Mean life span ± standard error: WT (N2), 16.8 ± 0.2 (386 animals); mek-1(ks54), 16.6 ± 0.4 (174 animals ) (P = 0.7089); unc-16(e109), 15.7 ± 0.3 (240 animals) (P = 0.0023).

Fig. 6. The jnk-1 transgenic worms show an increase in jnk-1 transcription level. The transcription level was measured in two independent integrated transgenic lines by RT-PCR (5'-ACAGTGGAACAGGAGGAGG, 3'-ATACGGAAGTGGAGGTGGAG) and compared with the WT. Data for each strain (% of N2): WT (N2), 100%; jnk-1, 0%; lpIn1, 1,368%; lpIn2, 1,432%.

Fig. 7. Overexpression of jnk-1 shows a further lifespan extension on akt-1/akt-2 RNAi. Life span analysis of jnk-1 overexpression strain (lpIn1) on akt-1/akt-2 RNAi. Mean life span ± standard error: N2 on control RNAi, 16.4 ± 0.4 days; N2 on akt-1/akt-2 RNAi, 18.3 ± 0.6 days; lpIn1 on control RNAi, 19.1 ± 0.6 days; lpIn1 on akt-1/akt-2 RNAi, 26.5 ± 0.7 days (P < 0.0001).

Fig. 8. The kinase-dead JNK-1 (APF) fails to phosphorylate DAF-16. We created kinase-dead JNK-1 construct by replacing TPY residues with APF and performed the in vitro kinase assay by using the N-terminal portion of DAF-16 as a substrate. The expression of protein was confirmed by immunoblotting with anti-Xpress antibody.

Fig. 9. jnk-1(gk7) and jkk-1(km2) are sensitive to heat stress. Fifty young adult worms were picked onto NGM plates and kept at 35°C. Animals were tapped every hour and scored as dead when they did not respond to the platinum wire pick. Mean survival time ± standard error: N₂, 13.9 ± 0.3 (90 animals); jnk-1(gk7), 13.5 ± 0.3 (86 animals); jkk-1(km2), 11.1 ± 0.2 (37 animals).

Fig. 10. jnk-1 overexpression strains increase resistance to oxidative stress. Approximately 30-40 young adults were transferred to 96-well plates (five to six worms per well) containing 40 m l of 150 mM paraquat at room temperature. Animals were tapped every 30 min and scored as dead when they did not respond to the platinum wire pick. Mean survival time ± standard error: control (N2 + pRF4), 1.7 ± 0.1 (88 animals); lpIn1, 3.1 ± 0.1 (68 animals); lpIn2, 2.7 ± 0.1 (118 animals). This stress assay was repeated at least three times.

Fig. 11. Heat stress resistance by jnk-1 overexpression depends on daf-16. Fifty young adult worms were grown and picked onto either control RNAi plates or daf-16 RNAi plates and kept at 35°C. Animals were tapped every hour and scored as dead when they did not respond to the platinum wire pick. Mean survival time ± standard error: N2 + pRF4 on control RNAi, 9.9 ± 0.2 (60 animals); N2 + pRF4 on daf-16 RNAi, 9.3 ± 0.1 (84 animals); lpIn1 on control RNAi, 12.0 ± 0.2 (98 animals); lpIn1 on daf-16 RNAi, 9.4 ± 0.1 (100 animals).