The mRNA levels of icl-1, adh-1, acdh-1, and hach-1 are dramatically adjusted upon fasting. (A) Schematic diagram to show how the fasting assay and the following imaged-based or qRT-PCR-based measurements were performed. (B) Measurement of mRNA abundance of icl-1, adh-1, acdh-1, and hach-1 in wild-type (N2) by quantitative RT-PCR after 8 h of fasting. The y-axis shows the mRNA abundance values normalized to well-fed WT. Worms were synchronized to the L4 stage before fasting treatment. The relative mRNA abundance for each gene was normalized to ama-1. n represents the number of biological replicates. (C) Representative images showing the localization of MCP::GFP with or without NLS. L4-stage worms were imaged. mCherry::LMN-1 labels the nuclear membrane. z-stacks were captured and proper single-focal-plane images were shown. Noted that NLS::MCP::GFP are likely enriched in the nucleoli. Scale bars: 5 μm. (D) Quantification of signal-to-noise ratio (SNR) for MCP::GFP with or without NLS. SNR was calculated as the ratio of the intensity of a fluorescent signal and the value of the background noise. n = 100 nuclei. (E) Schematic of the DualTag-based imaging in the epidermis of C. elegans. Dotted box: imaging site. (F) Schematic diagram of how to calculate the total RNA intensity transcribed in a nucleus and a locus (single allele). Green dots: nascent RNAs. (G) Quantification of total nascent transcripts of icl-1, adh-1, acdh-1, and hach-1 produced by each nucleus in the skin at different time points of fasting. Worms at the L4 stage were collected, treated with or without fasting, and imaged. n represents the number of nuclei. All values are displayed as mean ± SEM. ns: not significant. **P < 0.01, ****P < 0.0001 (one-way ANOVA with the Tukey correction).