Correlation of Hog1:GFP foci with mRNA-containing granules and the SG marker Puf2.
<p><b>A)</b> Detection of mRNA. <i>C. heterostrophus</i> cells expressing Hog1:GFP fusion protein were grown and prepared as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1013620#ppat.1013620.g001" target="_blank"...
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2025
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| Итог: | <p><b>A)</b> Detection of mRNA. <i>C. heterostrophus</i> cells expressing Hog1:GFP fusion protein were grown and prepared as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1013620#ppat.1013620.g001" target="_blank">Fig 1</a> and exposed to DMSO (control), 1.5 mM FA or 2.5 mM FA. Cells were fixed with 4% paraformaldehyde, permeabilized, probed with oligodT:Cy3 (smFISH; see Methods) and imaged (Spinning disk confocal, x100). The magenta channel is the FISH hybridization signal (Cy3), and the green channel is Hog1:GFP fluorescence. <i>Δhog1</i>, Hog1 deletion in the C4 background. Images are representative of three hybridization sets (see <b>B)</b>. Scale bars = 3 μm. <b>B)</b> Quantitation of overlap of Hog1:GFP and smFISH fluorescence signals. Spots were counted by Imaris software (for details see Methods) on 5-6 images from each of three hybridization sets on independent cultures (16 images total) and analysed for the number of smFISH signal (denoted parent, red) spots overlapping, containing or coinciding with Hog1:GFP spots, and inversely, the number of Hog1:GFP spots (denoted parent, green) overlapping, containing or coinciding with smFISH spots. Left, fraction of overlapping spots for each analysis direction plotted separately (“parent” denotes which channel was counted first). The fraction of overlapping spots among the total number of spots counted, calculated from the second analysis step and therefore independent of the direction of analysis, was 0.36 ± 0.07, mean and SEM of 16 images. Right panel, illustration of the spot analysis on a portion of a single image. First row: from left to right, green channel, Hog1:Gfp; red, smFISH; blue, DAPI. Second row: from left to right, all three channels; overlay, green and red channels; green and red channels, with the detected overlaps plotted as bright spheres. Scale bars 5 mm. <b>C)</b> Colocalization of the SG marker Puf2 with Hog1:Gfp foci. <i>C. heterostrophus</i> cells expressing both Hog1:GFP and Puf2:dTtomato fusion proteins were prepared and treated as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1013620#ppat.1013620.g001" target="_blank">Fig 1</a> except that the samples were not fixed, and live images were collected at 10 min intervals over 1 h. An image of Hog1:GFP and Puf2:dTtomato signals and their overlap at 60 min post FA induction is shown; representative of two independent experiments. Scale bars, 2 μm. For animations of the full time series see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1013620#ppat.1013620.s011" target="_blank">S1 Video</a> (DM) and 2 (FA). Hog1:GFP foci (green channel) are clearly visible by 20 min and Puf2 foci (red channel) around 40 min.</p> |
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