Rege-1 promotes C. elegans survival by modulating IIS and TOR pathways
Fig 3
The IIS pathway genes contributes to the PA14 sensitivity of rege1(imm070).
(A,B) PA14 survival curves of wild-type (WT), rege-1(imm070) and (A) daf-2(e1370) and rege-1(imm070);daf-2(e1370), (B) pqm-1(tm8184) and rege-1(imm070); pqm-1(tm8184). (C) Lifespan of wild-type (WT), rege-1(imm070), pqm-1(tm8184) and rege-1(imm070); pqm-1(tm8184) at 20°C. Three and two replicates have been performed in all the PA14 survival curves and lifespan, respectively. (D) Ratio of ETS-4::GFP nuclear localization in N2 and daf-16(mu86). The extrachromosomal array of ets-4::gfp was injected into either N2 or daf-16(mu86) strains, and the GFP intensity was quantified as described in the methods. Due to strong autofluorescence background, N2 worms without ets-4::gfp injection was also quantified as N2 without ETS-4::GFP. Worms with successful plasmid injection was scored by containing dominate negative rol-6 plasmid which shows a strong roller phenotype. The relative nuclear GFP intensity ratio is shown on the right. This ratio was calculated as the intensity of the nucleus divided by the intensity of the intestinal cell and was further normalized with the N2 strain without the ets-4::gfp plasmid injection. The white scale bars in the corner of the images represent a length of 10 μm. Two to three nuclei were quantified in each worm, and statistical significance between samples was determined using a one-way ANOVA with Turkey’s multiple comparison test. (E) Genome browser displaying ETS-4 ChIP-seq data on ins-7. The exon of the gene is denoted as a green box, the intron is shown as a black line, the orange line indicates the 3’ or 5’ UTR defined in the genome browser, and the black arrows indicate the direction of the gene. The blue lines below the gene structures represent the read intensity identified from ETS-4 ChIP-seq data. (F) Quantification of ins-7 mRNA in wild-type, rege-1(imm070), ets-4(ok165), and rege-1(imm070);ets-4(ok165) fed with OP50 or PA14. Three biological replicates were performed for each sample. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. (G) GFP expression was analyzed in pins-7::gfp strains in either N2 or rege-1(imm070) genetic backgrounds. Representative images are shown on the left, and quantification of GFP intensity is shown on the right. Significant differences between samples were calculated using an unpaired t-test. The scale bar in the corner of the image represents a length of 200 μm. Each experiment was scored with 15–20 worms, and two independent repeats were performed for each experiment. (*p < 0.05, **p < 0.001, ***p < 0.0002, ****p < 0.0001).