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. 2020 Feb 7;48(7):3888–3905. doi: 10.1093/nar/gkaa068

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Sequestration into ribosome-enriched foci involves both 40S and 60S-A501U subunits and is not a part of 60S-RQC. (A) BY4741 cells expressing indicated 25S^tag rRNAs were analyzed by RNA-FISH using probes against 18S^tag (Cy5-FL127) and 25S^tag (Cy5-FL128) rRNAs. Scale bar, 2 μm. (B) Percentage of cells with 25S^tag rRNA foci in cell populations transformed with the indicated mutant 25S rRNA constructs (n > 100 cells). Mean values and standard errors are shown, P values were determined by one-way ANOVA plus Dunnett's multiple comparisons test, NS is non-significant. (C) Cells were grown as in (A), and half of each culture was treated with 50 μM Bortezomib (BZ) for 1 h. 25S^tag rRNA was visualized by FISH with the Cy5-FL128 probe, while 18S^tag rRNA was visualized with the Atto 488-FL127 probe. An enlarged image for 18S^tag and 25S^tag-A501U rRNAs co-staining after BZ treatment is shown on the right. Yellow arrows point to the A501U-ribosome enriched foci. DAPI staining visualizes the nucleus (Nu). Representative microscopy fields are shown; scale bar, 2 μm. (D) Percentage of cells with 25S^tag rRNA foci in cell populations expressing the indicated 25S^tag rRNA constructs treated or untreated with BZ for 1 h (n > 100 cells). Mean values and standard errors are shown, P values were determined by one-way ANOVA plus Dunnett's multiple comparisons test. (E) pDP333 containing 25S^tag-A501U rRNA was transformed into wild-type BY4741, ltn1Δ and vms1Δ strains; transformants were grown as in (A), cells were analyzed by FISH with the Cy5-FL128 probe. Percentages of cell populations displaying 25S^tag rRNA foci were quantified for each strain tested (n>200 cells). Mean values and standard errors are shown, P values were determined by one-way ANOVA plus Dunnett's multiple comparisons test, NS is non-significant. (F) BY4741 cells containing 25S^tag-A501U rRNA were grown as in (A), one half of this culture was treated with 50 μg/ml cycloheximide (CHX) for 1 h prior to FISH with the Cy5-FL128 probe. Cells containing foci were quantified as in (E), >200 cells were analyzed. P value was determined by Mann Whitney test, NS is non-significant.