Figure 5 : MML-1/MXL-2 regulate HLH-30 via regulating TORC1 activity.

From: Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals

Figure 5

(a) Western blot analysis of phosphorylation of p70 S6 kinase. Bars show mean±s.e.m. of the band intensity relative to glp-1 from three experiments. The band intensity of p70 S6 kinase phosphorylation is normalized to b-actin. P-value (**P<0.01 and ***P<0.001) is determined by t-test relative to glp-1. (b) Bars show the percentage of worms with HLH-30::GFP in the nuclei of intestinal cells at day 1 adult (mean±s.e.m. from 3 biological replicates, >20 worms each). Each RNAi treatment is indicated. Knockdown of TORC1 but not TORC2 components rescues HLH-30 nuclear localization in glp-1mml-1 and glp-1mxl-2. Worms of individual genotypes were fed with indicated RNAi from egg onwards and raised at 25 °C. P-values (*P<0.05, **P<0.01 and ***P<0.001) were determined by t-test. (c) qRT–PCR analysis shows that lars-1 but not mars-1 is de-repressed in glp-1mml-1. The bar represents mean±s.e.m. from three biological replicates relative to N2. P-value (***P<0.001) was determined by one-way analysis of variance (ANOVA) with Tukey’s test relative to glp-1. (d) Representative fluorescent pictures of LARS-1::GFP. LARS-1::GFP is de-suppressed in glp-1mml-1 and glp-1mxl-2. (e) Quantification of LARS-1::GFP in posterior intestinal cells of indicated genotypes. Bars indicate mean±s.e.m. from 3 replicates (>20 worms each). P-value (***P<0.001) is determined by t-test relative to glp-1. (f) Representative fluorescent pictures of HLH-30::GFP in indicated genotypes and RNAi treatments at day 1. lars-1 knockdown rescues HLH-30 nuclear localization in double mutants. (g) Percentage of worms with HLH-30::GFP in the nuclei of intestinal cells at day 1 adult (mean±s.e.m. from 3 replicates, >20 worms each). Each RNAi treatment is indicated. (h) A representative western blotting showing phosphorylation of p70 S6 kinase and b-actin (left), and their quantification (right). lars-1 knockdown by RNAi significantly reduces the level of phosphorylation of p70 S6 kinase in glp-1mml-1 and glp-1mxl-2 background. The bar represents mean±s.e.m. from three experiments. P-value (*P<0.05 and **P<0.01) was determined by t-test. (i) mml-1 and mxl-2 are required for TOR knockdown-mediated longevity. TOR RNAi knockdown was conducted from the day 1 adult stage. Scale bars, 100 μm (d) and 50 μm (f).