Fig. 7: Differential recruitment of two different model RNAs tagged with Riboglow-FLIM to stress granules (SG) in live cells. | Nature Communications

Fig. 7: Differential recruitment of two different model RNAs tagged with Riboglow-FLIM to stress granules (SG) in live cells.

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

Fig. 7

a Cartoon illustration of simultaneously detecting subcellular localization of two RNAs via Riboglow RNA tags (A, D) that exhibit distinct lifetime values. b Representative images of arsenite-stressed HaloTag-G3BP1 U-2 OS cells simultaneously producing ACTB-Ribo(4D)−590 and 1/8-NORAD-Ribo(4A)−590. The Riboglow-tagged reporter was transfected together with a transfection marker, JF646 dye was added to mark the SG marker protein HaloTag-G3BP1, and the nucleus was labeled via NucBlue. Left: fluorescence intensity image showing SG and nucleus, and illustrating how SGs were identified (JF646-labeled SGs, yellow). Right: pixel-by-pixel component lifetime image. Dashed line with white arrow: ROI of SG. Solid line labelled with ‘C’: representative ROI of cytosol. Scale bar = 10 μm. c Average fluorescence lifetime (as defined in Fig. 2) value of ROIs representing SGs and cytosol, defined as an average value for the entire ROI, as illustrated in b. Lifetime values for live cells (21 cells, 42 ROIs, 2 independent experiments) listed in the box-whisker plot in which whiskers = minimum and maximum values, black line= mean, box represents interquartile range (25th percentile to 75th percentile). Dotted lines represent the mean of the lifetime for benchmarks established from ACTB-tagged mRNA in Fig. 4.

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