Fig. 6: Cholesterol activates mTORC1 at the lysosomes to boost growth during dauer exit. | Communications Biology

Fig. 6: Cholesterol activates mTORC1 at the lysosomes to boost growth during dauer exit.

From: Mobilization of cholesterol induces the transition from quiescence to growth in Caenorhabditis elegans through steroid hormone and mTOR signaling

Fig. 6

a Growth/length in mm in daf-15::mNeonGreen::AID;TIR1;daf-2 exiting dauers induced by temperature switch treated with 400 µM auxin (green) or solvent control (ethanol; black). Average and individual values of 3 independent experiments with a minimum of 30 worms per condition, two-way RM ANOVA with Geisser-Greenhouse correction showed p = 0.0018. b Survival of daf-15::mNeonGreen::AID;TIR1;daf-2 exiting dauers treated with auxin or solvent control. Average and individual values of one experiment with three biological replicates. Log-Rang analysis of the solvent control group compared to the auxin-treated group showed p < 0.0001. c Scheme of experimental procedure in (d). d daf-15::mNeonGreen::AID;TIR1 grown on 400 µM auxin and with cholesterol (+chol.) or without cholesterol (−chol.) until arrested as L3. Auxin was removed (0 h) and worms started re-growth on +chol. or –chol. Average and individual values of 2 independent experiments with a minimum of 30 worms per condition, two-way ANOVA with post hoc Tukey’s test comparing +/+chol with −/−chol showed p = 0.0007. e Growth/length in mm in daf-15::mNeonGreen::AID;TIR1;daf-2 exiting dauers on RNAi against rab-7, treated with 1 mM cholesterol (turquoise) or 13 µM cholesterol (black/orange) and with (green) or without 400 µM auxin (light green). Average and individual values of one experiment with a minimum of 29 worms per condition, two-way ANOVA with post hoc Tukey’s test comparing rab-7 with rab-7 high chol. showed p < 0.0001, rab-7 high chol. with rab-7 high chol. + auxin p < 0.0001. EV controls see Fig. S4h. f Growth/length in mm in daf-15::mNeonGreen::AID;TIR1;scl-12&13;daf-2 exiting dauers on 200 nM DA (red), auxin (green) and a combination of DA and auxin (light green), compared to solvent control. Average and individual values of one experiment with a minimum of 24 worms per condition, two-way ANOVA with post hoc Tukey’s test comparing Ctrl vs. DA showed p < 0.0001, Ctrl vs. auxin p < 0.0001, Ctrl vs. DA + auxin p < 0.0001, and DA vs. DA + auxin p < 0.0001. g Scheme of experimental procedure in (h). h daf-15::mNeonGreen::AID;TIR1 were grown without cholesterol until they were arrested in the F2 generation. Graph shows the percentage of the population that stays in the arrested state upon the addition of cholesterol (0%), 200 nM DA (red), 400 µM auxin (green), and cholesterol together with auxin (light green), compared to the solvent control. Average and individual values of three independent experiments with a minimum of 40 individual worms per condition. Only cholesterol is fully able to rescue the arrest (3 times an experimental value of 100% for Ctrl and 3 times 0% for cholesterol). An unpaired t-test comparing Ctrl and DA treatment showed p = 0.1497, and comparing cholesterol and DA p = 0.0002. i Exited daf-2 and scl-12&13;daf-2 worms/L4 after 48 h of induction of dauer exit treated with EV and nprl-3 RNAi (white). Average and individual values of 3 independent experiments with a minimum of 25 worms per condition each, unpaired two-tailed t-test between daf-2 EV and scl-12&13;daf-2 EV showed p = 0.0007 and between scl-12&13;daf-2 EV and scl-12&13;daf-2 nprl-3 p = 0.0005.

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