Validation of nascent RNA labeling. (A) Schematics to show the designs for nascent RNA and protein colabeling of endogenous genes in C. elegans. SL2, trans-spliced leader. (B) Schematics showing the dual imaging of nascent RNA and protein. Nascent RNAs are bound by MCP::GFP. Proteins of interest are fused with mCherry. (C) Representative images to show simultaneous visualization of RNA (left) and protein (middle) of icl-1 in the skin cells. BFP::LMN-1 labels the nuclear membrane. L4 stage worms were imaged with or without 8 h fasting treatment. (D) % of nucleus with RNA spots indicating transcription level of icl-1 under the condition of C. (E and F) Confocal images and quantifications to show adh-1 transcriptional response to fasting in the absence or presence of α-amanitin. (G and H) Representative images and quantifications of the transcriptional level of acdh-1 with or without α-amanitin treatment. (I and J) Fluorescent images and quantifications of transcriptional level of hach-1 in the presence or absence of α-amanitin. All images in Fig. 2 were captured by z-stacks and proper single-focal-plane images were shown. Arrowheads: nascent RNA. The boxed region is magnified (4×). Scale bars:5 μm. n represents the number of animals. All values are displayed as mean ± SEM. ns: not significant. ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t test).