Mobilization of cholesterol by 2-AG is dependent of <i>hid-1</i>.

(A) Hid-1 is required for 2-AG dependent mobilization of cholesterol. Worms were grown at 27°C under normal dietary cholesterol. All pairwise are multiple comparison procedures (Holm-Sidak method), *p hid-1 background restores the 2-AG-dependent mobilization of cholesterol. Worms were grown at 27°C under normal dietary cholesterol. Mann-Whitney rank sum test, *p (TIF)</p

Dauer percentage in RNAi enhancer screen essays on <i>daf-7 (e1372)</i> worms.

For details see text. (DOCX)</p

Adult percentage in orthologues of CB1/2 mutants grown for two generations in the absence of cholesterol arrest as L2* larvae.

(DOCX)</p

DAF-2 and UNC-31 are required for 2-AG-dependent cholesterol mobilization.

(A) daf-7 and daf-2(e1370) were grown at 20°C either in cholesterol or in a sterol-free media during one generation. When indicated the media was supplemented with either 13 μM cholesterol or 50 μM 2-AG. All Pairwise are Multiple Comparison Procedures (Dunn’s Method), *p age-1 were grown for two generations in media with 0 μg/ml cholesterol at 20°C. Mann-Whitney rank sum test, *p akt-1 was grown in media with 0 μg/ml cholesterol during one generation at 25°C. All values are from n = 3 independent experiments shown as Mean ± SEM. ns = not significant. (D) 2-AG is unable rescue the daf-c phenotype of unc-31 grown at 27°C. All Pairwise are Multiple Comparison Procedures (Dunn’s Method), *p C. elegans wild-type strain.</p

FAAH-4 is involved in cholesterol mobilization by 2-AG.

(A) Wild type and faah-4 animals grown for two generations in the absence of cholesterol at 20°C arrest as L2*. Feeding with 2-AG (50 μg/ml) increases the formation of adults in faah-4 animals compared with N2 animals. All Pairwise are Multiple Comparison Procedures (Dunn’s Method), *p faah-4 animals. T-test, *p faah-4 RNAi reduces the dauer formation of strain ncr-2; ncr-1 and enhances the ability of low concentrations of 2-AG (10 μM) to suppress the daf-c phenotype of these animals. All Pairwise are Multiple Comparison Procedures (Holm-Sidak method), *p C. elegans wild-type strain.</p

The cannabinoid receptor antagonist NMP331 induces dauer formation.

(A) The dauer arrest of daf-7 worms is augmented by NMP331. All pairwise are multiple comparison procedures (Holm-Sidak method), *p fat-4 animals undergo a dauer-like arrest induced by NMP331 in the first generation when grown in cholesterol free medium. Mann-Whitney rank sum test, *p C. elegans wild-type strain.</p

TPH-1 is not required for 2-AG-dependent cholesterol mobilization.

(A) N2 and tph-1 were grown for two generations in media with 0 μg/ml cholesterol at 20°C. Mann-Whitney rank sum test, *p C. elegans wild-type strain. (TIF)</p

Endocannabinoids stimulate transport of cholesterol by employing pathways independent of <i>npr-19</i> and <i>npr-32</i>.

(A) 2-AG suppresses L2* larval arrest of npr-19, npr-32 and npr-19; npr-32 animals grown for two generations on plates containing 0 μg/ml of cholesterol. All values are from n = 3 independent experiments shown as Mean ± SEM. ns = not significant. N2 is the C. elegans wild-type strain. (B) Endocannabinoids suppress dauer arrest of daf-7 and daf-7; npr-19 grown on plates containing 5 μg/ml of cholesterol. Mann-Whitney rank sum test, *p < 0.001, **p < 0.001. All values are from n = 3 independent experiments are shown as Mean ± SEM. The concentration of 2-AG was 50 μM and of 2-AGE 100 μM.</p

Mobilization of cholesterol by 2-AG is independent of the <i>daf-7</i> pathway.

(A). Daf-4 was grown at 20°C while daf-14 and daf-8 were grown at 25°C under normal dietary cholesterol (13 μM). t-test, *p daf-7 and daf-2 were grown at 23°C in a sterol-free media during one generation. t-test, *p daf-2 mutants. Mann-Whitney rank sum test, *p (TIF)</p

ASH calcium levels are not affected by 2-AG.

Calcium imaging traces of animals expressing GCaMP6 in the ASH (xuEx1978 [Psra-6::Gcamp6(f), Psra-6::DsRed]) during exposure to 2-AG. Average responses for all animals (A) are indicated by the dark blue line and the shaded area represents the SEM. The same results are shown as individual traces for each animal (B). The dark grey bars indicate 4 second exposure to buffer containing 100 mM 2-AG. (TIF)</p