For qRT-PCR analysis of food-deprived worms, wild-type embryos were obtained by using a bleaching method (7) and placed on control RNAi or vrk-1 RNAi bacteria-seeded NGM plates. Worms were then grown until reaching day 2 adults, harvested with M9 buffer, and washed three times to remove remaining bacteria. For a fed condition, worms were placed on control RNAi bacteria– or vrk-1 RNAi bacteria–seeded NGM plates. For a food deprivation condition, worms were placed on NGM plates containing streptomycin (10 μg/ml) to prevent E. coli (HT115) that expresses dsRNA from growth, harvested with M9 buffer, and used for RNA extraction and qRT-PCR analysis after 24 hours of food deprivation. FUDR was applied to prevent progeny from hatching at L4 stage. For assaying the subcellular localization of CRTC-1::RFP, worms were treated with control RNAi from hatching until they reached L2 or L3 stages. The animals were then collected with M9 buffer, washed three times, and placed on NGM plates containing streptomycin (50 μg/ml) and kanamycin (50 μg/ml) for food deprivation. After 24 hours of food deprivation, the worms were used for micrograph imaging.

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