Figure 6: The PKD homologue DKF-2 acts downstream of G protein-PLCβ signalling but upstream of DAF-16 to regulate lifespan.

(a) dkf-2 acts in the gbb-1 pathway to regulate lifespan. dkf-2 mutant worms were long-lived (log-rank test, P<0.001). dkf-2; gbb-1 double mutant showed a similar lifespan to single mutants (log-rank test, P=0.919), suggesting that they are in the same pathway (n=61–72 for different genotypes). (b) Loss of daf-16 fully suppresses the long-lived phenotype of dkf-2 mutant worms (log-rank test, P=0.114, n=72–82 for different genotypes). (c) qPCR analysis of DAF-16 target genes. qPCR reactions were run in triplicates for each genotype. Each experiment was repeated three times. Error bars, s.e.m. *P<0.05 (analysis of variance (ANOVA) with Bonferroni test). (d) Quantification of SOD-3::GFP fluorescence intensity. SOD-3::GFP is encoded by the transgene muIs84 (ref. 46). The left panels show representative images (selected from 10 similar images). Image quantification was performed as previously described⁵⁶. n≥15. Error bars, s.e.m. **P<0.001 (t-test). Scale bar, 100 μm. (e) Overexpression of dkf-2 in neurons fully suppresses the long-lived phenotype of dkf-2 mutant worms. Prgef-1 is a neuron-specific promoter⁶² (log-rank test, P=0.143, n=77–98 for different genotypes). (f) Overexpression of dkf-2 modestly shortens lifespan (log-rank test, P=0.005, n=58–69 for different genotypes). (g) Overexpression of dkf-2 fully suppresses the long-lived phenotype of gbb-1 mutant worms (log-rank test, P=0.462, n=44–72 for different genotypes). (h) Overexpression of dkf-2 fully suppresses the long-lived phenotype of egl-8 mutant worms (log-rank test, P=0.967, n=50–98 for different genotypes). All lifespan assays were carried out at 20 °C and were repeated at least twice. FUDR was included in assays involving egl-8 mutant worms, which show a defect in egg laying. Please see Supplementary Table 1 for detailed statistical analysis of lifespan data.