<i>hlh-30</i> was necessary for survival during starvation and upon refeeding, and starvation promotes HLH-30 nuclear localization.

(A) Schematic depicting starvation and refeeding assay. Gravid adults were bleached with alkaline hypochlorite to obtain eggs, which were transferred to buffered salt solution (M9). Upon hatching, larvae arrest development at the L1 stage. Aliquots of starved L1 worms were placed on NGM dishes seeded with E. coli (OP50), and worms were immediately scored as alive or dead based on spontaneous movement; the fraction alive were defined as “Alive After Starvation.” After two days, worms were again scored as alive or dead based on spontaneous movement; the fraction alive compared to two days previously were defined as “Alive After Refeeding.” Because “Alive After Starvation” and “Alive After Refeeding” are calculated using different denominators, either value may be higher. (B, C) WT and hlh-30(lf) worms were analyzed after variable periods of starvation (N = 4 biological replicates with 50 worms/time point; values indicate mean ± SEM). *P hlh-30(oe) or to hlh-30(mNLS)(oe) were analyzed at the L1 stage in the fed state or after 33 hours of starvation. Protein extracts were separated to obtain cyto and nuc fractions. Representative immunoblots (above) illustrate protein levels, and quantification of HLH-30 levels in the cytosol (normalized to GAPDH levels, fed state set equal to 1.0) and nucleus (normalized to Histone H3 levels, fed state set equal to 1.0) are shown below. N = 3 biological replicates/group. Bars indicate mean ± SEM. *P t test. Lanes 1 and 2 of the immunoblot in panel D contain extracts from WT worms lacking RFP expression and document the specificity of RFP detection. (F) WT, hlh-30(lf), hlh-30(oe), and hlh-30(mNLS)(oe) worms were analyzed after 33 hours of starvation for “Alive after Refeeding.” N = 3 biological replicates/group of approximately 50 worms. Bars indicate mean ± SEM. *P S1 Data. cyto, cytoplasmic; GAPDH, glyceraldehyde 3-phosphase dehydrogenase; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(mNLS)(oe), overexpressed HLH-30 with a mutant nuclear localization signal; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; hlh-30(oe), overexpressed HLH-30; L1, first larval stage; NGM, nematode growth medium; nuc, nuclear; RFP, red fluorescent protein; SEM, standard error of the mean; WT, wild type.