NHR-49 and HLH-30 act as transcription factors to upregulate icl-1 and adh-1 expression upon fasting. (A) Schematic diagram of DualTag-based imaging system to detect the endogenous icl-1 transcriptional level. (B) Schematic diagram of the potential dynamic transcriptional changes of icl-1 or adh-1 induced by fasting. Green dots: nascent RNAs labeled by MCP::GFP. (C–E) Quantification of the percentage of nuclei with RNA spots, number of transcriptional active alleles per nucleus, intensity of single RNA spot, and total intensity of all alleles per nucleus of icl-1 in WT and nhr-49(zac598) (C), hlh-30(tm1978) (D), or daf-16(mu86) (E). nhr-49(zac598), hlh-30(tm1978), and daf-16(mu86) are deletion mutants, resulting in strong loss-of-function or null alleles. For analyzing the % of nuclei with RNA spots, n represents the number of animals. For quantifying the intensity of a single RNA spot, n represents the number of alleles. n represents the number of nuclei when analyzing the number of transcriptional active alleles per nucleus and total intensity of all alleles per nucleus. The exact numbers analyzed were indicated above each bar. (F–H) Similar to C–E except that adh-1 was analyzed. All values are displayed as mean ± SEM. ns: not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (comparison between multiple groups with the same genotype: one-way ANOVA with the Tukey correction; comparison between two groups with different genotypes: two-tailed unpaired Student’s t test).