DualTag-based imaging sensitively reports the transcriptional expression level of hsp-4/BiP upon ER stress. (A) Representative images showing that hsp-4p::GFP reporter responses to ER stress (caused by tunicamycin treatment for different durations). L4 stage worms were imaged. All images are maximum intensity projections from z-stacks. Scale bars: 200 μm. (B) Representative images illustrating the level of the nascent transcripts of the endogenous hsp-4 induced by ER stress. Z-stacks were captured and proper single-focal-plane images were shown. Scale bars: 20 μm. Arrowheads: nascent RNAs. (C) Quantification of mean fluorescence intensity of each worm under different conditions as shown in A and % of the nucleus with RNA spots as shown in B. n represents the number of animals. (D) Quantification of individual spot fluorescence, number of active alleles per cell and total intensity of all spots per nucleus under different conditions as shown in B. When quantifying the intensity of single RNA spot, n represents the number of alleles. n represents the number of nuclei for quantifying the number of transcriptional active alleles per nucleus and transcriptional output of single nuclei. The exact numbers analyzed were indicated above each bar. All values are displayed as mean ± SEM. ns: not significant. ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t test).