Fig. 6: Quantification of RNA recruitment to stress granules (SGs) for different tagged RNAs via Riboglow-FLIM. | Nature Communications

Fig. 6: Quantification of RNA recruitment to stress granules (SGs) for different tagged RNAs via Riboglow-FLIM.

From: Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM)

Fig. 6

a Representative images of arsenite-stressed HaloTag-G3BP1 U-2 OS cells producing truncated versions of Riboglow-tagged NORAD. Cells produce the SG marker protein G3BP1 from the chromosome to identify SGs via HaloTag ligand JF646 (top panel, fluorescence intensity image, yellow areas indicate G3BP1). Nucleus was identified using NucBlue (blue). White arrows point to HaloTag-G3BP1 marked SGs. Smaller white circle labeled C indicates a representative ROI of the cytosol. Bottom panel: pixel-by-pixel component lifetime image of the same field of view visualized on a false color scale. Scale bar = 10 μm. b The average fluorescence lifetime value was determined where each dot corresponds to an ROI representing the whole cell, or a SG (circles, indicated by white arrow), or a randomly selected ROI of similar area in the cytosol (‘C’) (23 cells, 100 ROIs, 8 total independent experiments, 1 symbol = 1 ROI). Dotted lines represent the mean of the lifetime for benchmarks established from ACTB tagged mRNA in Fig. 3. One-way ANOVA (95% confidence limit); post hoc test (Tukey HSD). Error bars indicate mean and standard deviation (+/−SD).

Back to article page