Preparation of conditioned media

Hermaphrodite- and male-secreted metabolites were collected according to a previous publication (Srinivasan et al., 2008). Synchronized C. elegans (WT [N2], him-5 [N2], WT [TR389], him-5 [TR389], daf-22 [N2], and daf-22; him-5 [N2]) with a density of 10,000 worms/plates (90 mm, three plates) were grown on the nematode growth media (NGM) agarose (seeded with E. coli strain OP50) at 20°C. There were 43.07 ± 0.77%, 39.26 ± 1.55%, and 37.29 ± 1.28% males in him-5 (N2), daf-22; him-5 (N2), and him-5 (TR389) strains, respectively. After worms reached the young adult stages, they were collected and washed three times with M9 buffer to remove bacteria. To further remove the bacteria in the gut, the worms were then placed in M9 buffer in a shaker (150 rpm) at 20°C for 30 min and rinsed three times with ddH₂O. Subsequently, worm-secreted metabolites were collected by incubating the worms in ddH₂O in a shaker (150 rpm) for 3 hr with a density of 30,000 worms/ml. Afterward, the worms were removed by settling on ice for 5 min. The metabolites were filtered through 0.22 μm filters, aliquoted, and stored at −80°C. For conditioned medium preparation, 10 μl metabolites mixed with 90 μl OP50 E. coli were spread on a 35 mm NGM plate. Plates were allowed to dry overnight before use.