A) akt-1;akt-2 or sgk-1 RNAi allows daf-16-independent accumulation of SKN-1 in intestinal nuclei. Expression of SKN-1op::GFP was monitored. akt-1 or akt-2 RNAi had a similar effect to combined akt-1;akt-2 RNAi (data not shown). B) RNAi depletion of the downstream IIS kinases causes sek-1-dependent accumulation of SKN-1 in intestinal nuclei. Expression of SKN-1B/C::GFP was monitored. C) SKN-1C is phosphorylated by AKT-1, AKT-2, and SGK-1 comparably to DAF-16, dependent upon daf-2. Here and in panels (D–G), AKT-1::GFP, AKT-2::GFP and SGK-1::GFP were immunoprecipitated from WT or daf-2(e1370) C. elegans lysates with anti-GFP antibody, quantitated by Western blotting with anti-GFP, and used to phosphorylate purified GST-fused SKN-1 or DAF-16 proteins with [γP32]ATP. GST controls are shown in Figure S4B. These kinases appropriately required activation in C. elegans, as they did not phosphorylate SKN-1 after they had been expressed in E. coli (Figure S4A). D) The unique 90aa region at the SKN-A N-terminus (SKN-1A(1–90)) is phosphorylated by the AKT kinases but not SGK-1, dependent upon daf-2. E) Partial mapping of AKT-1 sites within SKN-1A(1–90). F) AKT and SGK phosphorylate particular regions of SKN-1C. Here and in panel (G), residues are numbered as in SKN-1A, so that the first residue of SKN-1C is numbered as 91. G) Partial mapping of AKT-1, AKT-2, and SGK-1 sites within SKN-1C fragments. H and I) Substitution of a strongly predicted AKT site within SKN-1A(1–90), Ser-12 (Table S1), results in nuclear accumulation of SKN-1A::GFP. In some lines low levels of SKN-1::GFP were observed in additional head neurons, hypodermis, and other tissues, particularly if Ser-12 was mutated (data not shown).

A) skn-1 contributes to the oxidative stress resistance phenotype of daf-2(e1370) mutants. A representative experiment is shown in which animals were exposed to 7.5 mM t-butyl hydrogen peroxide. B) skn-1 contributes to the stress resistance phenotypes conferred by sgk-1 or akt-1;akt-2 RNAi. Animals were scored after 5 days exposure to 150mM paraquat. Data were combined from at least three experiments with n>90 for each group. C) Lifespan analysis of daf-2(e1370); skn-1 mutants. D) skn-1 suppresses the longevity of daf-2(e1368) mutants. E) In the RNAi-sensitive rrf-3 background (Simmer et al., 2002), the lifespan increase seen in daf-2(e1368) is comparably suppressed by either skn-1 or skn-1a/c RNAi. RNAi bacteria were provided from the L1 stage. F, G) skn-1 mutations suppress the lifespan extension conferred by akt-1;akt-2 or sgk-1 RNAi. Survival assays were carried out at 25°C, are from adulthood, and represent the combined data from at least two experiments. H) skn-1 mutation does not suppress the lifespan extension conferred by cyc-1 RNAi. The lifespan experiments in C and D were carried out on OP50, but all others were performed on HT115 lawns. Lifespan data in this Figure are summarized in Table 1. Unless otherwise indicated, the analyses shown were performed at 20°C, and measured from hatching.

A) A partial schematic of C. elegans insulin-like signaling and our working model. B) skn-1 isoforms, alleles, and transgenes. The SKN-1A isoform is transcribed from an upstream operon promoter, and differs from SKN-1C by the addition of 90 amino acids at its N-terminus. SKN-1B and SKN-1C are each expressed from their own distinct upstream promoter (An and Blackwell, 2003; Bishop and Guarente, 2007). The skn-1(zu67), (zu129), and (zu135) mutations each create a premature stop codon (B. Bowerman, personal communication). Green Fluorescent Protein (GFP) is fused to the C-terminus of SKN-1 in the transgenes indicated at the bottom. Strains that carry these and other transgenes are described in Table S2. The skn-1a/c RNAi construct includes only coding sequence, and targets both SKN-1A and SKN-1C, but does not alter the levels of SKN-1B in the ASI neurons (Bishop and Guarente, 2007). C) SKN-1::GFP accumulates in intestinal nuclei when DAF-2 activity is reduced. D) Intestinal expression of SKN-1B/C::GFP in daf-2(e1370) derives from SKN-1C. E) Quantification of SKN-1 intestinal nuclear accumulation. Here and in Figure 2, results of experiments performed on L4 larvae have been combined and scored as described in the supplementary methods and in (An and Blackwell, 2003; An et al., 2005). p values were derived from a chi² test.

A and B) Schematics of the gcs-1 promoter (An and Blackwell, 2003), and the GST- 7::GFP translational fusion. Triangles mark SKN-1 binding sites. gcs-1Δ2::GFP lacks a promoter region required for SKN-1-independent pharyngeal expression, and gcs-1 Δ2mut3::GFP lacks a SKN-1 binding site that is important for skn-1-dependent gcs-1 expression in the intestine and ASI neurons (An and Blackwell, 2003). C) gcs-1::GFP is induced in the intestine independently of daf-16 in response to akt-1;akt-2 and sgk-1 RNAi. Here and in (D), results of multiple experiments performed on L4 larvae have been combined and scored as described in the supplementary methods and (An and Blackwell, 2003; An et al., 2005). p values were derived from a chi² test. akt-1 and akt-2 RNAi were also performed separately, and shown to have a similar effect to combined akt-1;akt-2 RNAi (data not shown). D) GST-7::GFP is induced in response to daf-2, akt-1;akt-2, or sgk-1 RNAi. E–J) SKN-1- and DAF-16-responsive genes that are upregulated in daf-2(e1370) L4 larvae were identified by quantitative PCR, using actin (act-1) as a control. The level of skn-1 mRNA in these samples was also measured (Figure S6). One representative experiment is shown, error bars represent the SEM, and p values were derived from a Student’s t-test.

A) Lifespan extension in otherwise WT animals carrying the [SKN-1B/C S393A::GFP] transgene, which expresses constitutively nuclear SKN-1C in the intestine (see text). These independent extrachromosomal arrays were created by injecting 2.5ng/µl transgene DNA. B) Lifespan extension in skn-1(zu67) animals carrying skn-1 transgenes. The Ex001[SKN-1B/C::GFP], Ex008[SKN-1B/C S393A::GFP] and Ex020[SKN-1B/C S393A::GFP] extrachromosomal arrays were originally created in WT animals by injection of 2.5, 2.5, and 10 ng/µl transgene DNA, respectively, and then bred into skn-1(zu67). The presence of the transgene did not dramatically affect the developmental timing, brood size or age specific fecundity of either the WT or skn-1 mutants (Figure S14). The lifespan of WT animals carrying Ex001[SKN-1B/C::GFP] is shown for comparison. C) SKN-1C contributes to the lifespan of skn-1(zu67) Ex001[SKN-1B/C::GFP] animals. Lifespan is reduced comparably by skn-1 and skn-1a/c RNAi (Figure 1B). D) Presence of Ex008[SKN-1B/C S393A::GFP] increases the lifespan of daf-16(mgDf47) mutants. E) Transgene arrays that express constitutively nuclear SKN-1 (Ex008 or Ex020) increase skn-1(zu67) lifespan beyond WT when daf-16 is depleted by RNAi. Lifespan analyses were scored from hatching and performed at 20°C unless otherwise stated, and are summarized in Table 2. The experiments in panels A and B were carried out on OP50, and the others on HT115. Panels C and D are representative experiments, but otherwise data were combined from at least two experiments, within which all sample sets were analyzed in parallel. F) Cross talk among C. elegans transcriptional regulators that increase longevity when expressed transgenically. DAF-16 physically interacts with SIR-2.1 through 14-3-3 proteins, and is required for SIR-2.1 to extend lifespan (Berdichevsky et al., 2006; Tissenbaum and Guarente, 2001). HSF-1 and DAF-16 are predicted to bind to some of the same promoters, and regulate some pro-longevity target genes together (Hsu et al., 2003). PHA-4 shares a common cooperating cofactor with DAF-16 (SMK-1), extends lifespan when DAF-16 is absent, and is required for DR to extend lifespan (Panowski et al., 2007). SKN-1 and DAF-16 are inhibited directly and in parallel by IIS (this work).