(A) Schematic representation of the Lnk locus along with the flanking genes CG17370, CG5913 and CG5948. The three Lnk transcripts (RA, RB, and RC) are shown along with the positions of the P-element insertion d07478 and the deletion Del29. Arrows show the position of primers used for the quantitative RT–PCR in (B). (B) Quantitative RT–PCR analysis of Lnk transcript expression in 7-day old female flies. Lnk transcripts were amplified using the primers indicated in (A) and normalised to actin5C. (C) Mean wing area and body mass for male and female flies of the indicated genotypes. Data are represented as means ± SEM (n = 10 for each measurement). Percentage differences compared to w¹¹¹⁸ controls are indicated (n.s.  =  not significantly different). (D) The wings of homozygous Lnk mutant females contain fewer and smaller cells compared to the w¹¹¹⁸ controls. Data are shown as means ±SEM (n = 10). ** denotes statistically significant difference (p<0.05).

(A) Survival of male flies fed 5% hydrogen peroxide. Lnk mutant males live approximately 50% longer than controls and mutants carrying a Lnk genomic rescue (GR) construct (p<0.001). Median survival times are 2.6 days for w^Dah (black line; n = 97), 3.9 days for Lnk^Del29 (blue line; n = 93) and 2.6 days for LnkGR; Lnk^Del29/Lnk^Del29 (green line; n = 100). (B) Survival of female flies fed 5% hydrogen peroxide. Lnk mutant females live approximately 50% longer than controls and mutants carrying a Lnk genomic rescue (GR) construct (p<0.001). Median survival times are 3.1 days for w^Dah (black line; n = 93), 5.6 days for Lnk^Del29 (red line; n = 105) and 3.1 days for LnkGR; Lnk^Del29/Lnk^Del29 (green line; n = 100). (C) Survival of male flies under starvation conditions. Lnk mutant males live 30% longer than controls and mutants carrying a Lnk genomic rescue (GR) construct (p<0.001). Median survival times are as follows: 2.0 days for w^Dah (black line; n = 92), 2.6 days for Lnk^Del29 (blue line; n = 86) and 1.9 days for LnkGR; Lnk^Del29/Lnk^Del29(green line; n = 97). (D) Survival of female flies under starvation conditions. Lnk mutant females live 35% longer than controls and mutants carrying a Lnk genomic rescue (GR) construct (p<0.001). Median survival times are as follows: 4.3 days for w^Dah (black line; n = 94), 5.8 days for Lnk^Del29 (red line; n = 95) and 4.7 days for LnkGR; Lnk^Del29/Lnk^Del29 (green line; n = 95). (E) Whole-fly content of triglycerides (TAG) per mg of fly (fresh weight). Data are presented as means (n = 10) ± SEM. ** denotes statistically significant difference (p<0.05). (F) Whole-fly glycogen content per mg of fly (fresh weight). Data are presented as means (n = 10) ± SEM. ** denotes statistically significant difference (p<0.05). (G) Whole-fly trehalose content per mg of fly (fresh weight). Data are presented as means (n = 10) ± SEM. ** denotes statistically significant difference (p<0.05).

(A) Schematic diagram showing in vivo binding region for dFoxo at the Lnk locus as detected by chromatin immunoprecipitation (ChIP). Transcriptional start sites and the direction of transcription are indicated by arrows. The genomic region 3′ to the Lnk locus is also marked. (B) Quantitative PCR (qPCR) on the Lnk promoter, a 3′ region of the Lnk gene [as highlighted in (A)], the known dFoxo target gene, Lk6 and the U6 snRNA promoter (-87 to +61 from transcriptional start) to determine the proportion of DNA recovered after ChIP using anti-dFoxo antibody under normal, starvation and paraquat-treated conditions. Relative DNA binding was calculated as the proportion of chromatin recovered in the ChIP divided by that in the total chromatin preparation. Data are presented as mean fold-changes in relative DNA–binding compared to U6 ± SEM of three biological repeats (** p<0.05). (C) Lnk mRNA levels in dFoxo mutant flies measured by qRT–PCR and normalised to actin5C. Data are represented as mean normalised transcript level ± SEM (n = 3; **p<0.05).

(A) w¹¹¹⁸-backcrossed females. Median lifespans are: 57 days for w¹¹¹⁸ (n = 196), 58 days for Lnk^d07478/+ (n = 203) and 65 days for Lnk^d07478/Lnk^d07478 (n = 163). Log rank test χ² and p-values: w¹¹¹⁸ versus Lnk^d07478/+ (χ² = 1.7, p = 0.192) and w¹¹¹⁸ versus Lnk^d07478/Lnk^d07478 (χ² = 90.2 , p<0.0001). (B) w¹¹¹⁸-backcrossed females. Median lifespans are 61 days for w¹¹¹⁸ (n = 173), 59 days for Lnk^Del29/+ (n = 200) and 68 days for Lnk^Del29/Lnk^Del29 (n = 182). Log rank test χ² and p-values: w¹¹¹⁸ versus Lnk^Del29/+ (χ² = 2.2, p = 0.139) and w¹¹¹⁸ versus Lnk^Del29/Lnk^Del29 (χ² = 65.2, p<0.0001). (C) w^Dah-backcrossed females. Median lifespans are 63 days for w^Dah (n = 179), 66 days for Lnk^Del29/+ (n = 177) and 68 days for Lnk^Del29/Lnk^Del29 (n = 178). Log rank test χ² and p-values: w^Dah versus Lnk^Del29/+ (χ² = 4.67, p = 0.03) and w^Dah versus Lnk^Del29/Lnk^Del29 (χ² = 53.09, p<0.0001). (D) Reduced fecundity in Lnk homozygous mutant females. Index of lifetime fecundity represents the mean number of eggs laid per female per day at 10 time points during the first 40 days of life. Index of lifetime fecundity in Lnk homozygous mutant females (white bars) is significantly reduced compared to control flies (w1118; dark grey bars) and heterozygotes (light grey bars). Data are shown as means ±SEM. ** denotes statistically significant difference (p<0.05). (E) w¹¹¹⁸-backcrossed males. Median lifespans are 57 days for w¹¹¹⁸ (n = 198), 57 days for Lnk^d07478/+ (n = 196) and 67 days for Lnk^d07478/Lnk^d07478 (n = 190). Log rank test χ² and p-values: w¹¹¹⁸ versus Lnk^d07478/+ (χ² = 0.1 , p = 0.778) and w¹¹¹⁸ versus Lnk^d07478/Lnk^d07478 (χ² = 107.0, p<0.0001). (F) w¹¹¹⁸-backcrossed males. Median lifespans are 63 days for w¹¹¹⁸ (n = 192), 63 days for Lnk^Del29/+ (n = 181) and 71 days for Lnk^Del29/Lnk^Del29 (n = 144). Log rank test χ² and p-values: w¹¹¹⁸ versus Lnk^Del29/+ (χ² = 0.5, p = 0.463) and w¹¹¹⁸ versus Lnk^Del29/Lnk^Del29 (χ² = 37.0, p<0.0001). (G) w^Dah-backcrossed males. Median lifespans are 52 days for w^Dah (n = 169), 52 days for Lnk^Del29/+ (n = 168) and 61 days for Lnk^Del29/Lnk^Del29 (n = 171). Log rank test χ² and p-values: w^Dah versus Lnk^Del29/+ (χ² = 0.24, p = 0.627) and w^Dah versus Lnk^Del29/Lnk^Del29 (χ² = 44.89, p<0.0001). (H) Mean number of eggs laid per w¹¹¹⁸ female crossed to males of the indicated genotype. Data are shown as mean number of eggs laid per female fly over a four day period ± SEM. (I) Genomic rescue (GR) of Lnk in w^Dah-backcrossed females. Median lifespans are 67 days for w^Dah (n = 151), 74 days for Lnk^Del29/Lnk^Del29 (n = 150) and 66 days for LnkGR;Lnk^Del29/Lnk^Del29 (n = 92). Log rank test χ² and p-values: w^Dah versus Lnk^Del29/Lnk^Del29 (χ² = 33.87, p<0.0001) and w^Dah versus. LnkGR;Lnk^Del29/Lnk^Del29 (χ² = 0.71, p = 0.40). (J) Genomic rescue of female fertility defects. Eggs were counted on day 7 of the lifespan experiment shown in (I). Data are shown as mean number of eggs/female over a 24 hour period ± SEM. ** denotes statistically significant difference (p<0.05).

(A) Heat map showing expression of genes within the IIS and Ras/MapK signal transduction pathways that were significantly altered in Lnk mutants compared to controls. Red indicates lower expression; blue indicates higher expression (scale  =  log2 fold change). (B) Transcript levels of six IIS pathway genes measured by qRT-PCR and normalised to actin5C. RNA was extracted from adult female heads. Data are represented as mean normalised transcript level ± SEM (n = 4; ** p<0.05). (C) 4eBP and dInR mRNA levels measured by qRT-PCR and normalised to actin5C. RNA was extracted from either heads or bodies of adult females. Data are represented as mean normalised transcript level ± SEM (n = 4; ** p<0.05).

Western blot analysis of Akt and Erk-A phosphorylation in protein extracts of Drosophila S2 cells after treatment with the indicated dsRNAs before (-) and after (+) stimulation with human insulin. Knockdown of dInR, chico, or Lnk expression inhibits both Akt and Erk-A phosphorylation after insulin stimulation whereas knockdown of Shc inhibits phosphorylation of Erk-A only consistent with its role as an adapter for Ras/MAPK signaling. dsRNA against GFP was used as a negative control. Blots were probed with anti-tubulin as a loading control. Knockdowns of Lnk and chico transcripts were confirmed by Northern blot; knockdowns of dInR and Shc expression were confirmed by western blot (data not shown).