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. 2017 Nov 20;46(3):1139–1156. doi: 10.1093/nar/gkx1160

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© The Author(s) 2017. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Precursor HAC1 mRNA is recruited more efficiently to Ire1p foci in DRN deficient cbc1-Δ strains due to the diminished decay of its 3′-BE. (A) Localization of Ire1p-RFP and HAC1^NRE mRNA decorated with NRE-GFP following the induction of ER stress by 1 μg/ml tunicamycin in a wild type and cbc1-Δ strain. The images were captured and processed as described in Materials and methods. (B) Histogram depicting the co-localization of HAC1^NRE-GFP-nucleolin mRNA into Ire1p-RFP foci at 10 minutes after ER stress by 1 μg/ml tunicamycin in a wild type and cbc1-Δ strain. Co-localization index for HAC1^NRE-GFP-nucleolin recruitment into Ire1 foci was expressed in arbitrary units. Means and standard error of the mean were determined from n = 17. Co-localization index (Pearson correlation coefficient, PCC) were determined as described in the method section. (C) Relative fluorescence resonance energy transfer (FRET) between HAC1-GFP-nucleolin and Ire1p-RFP in a wild type and cbc1-Δ strain. The bottom panel shows the quenching of donor HAC1- GFP-nucleolin mRNA in the presence of Ire1p-RFP (acceptor) in normal and cbc1-Δ strains. The top panel is the negative control of this experiment showing no donor (HAC1-GFP-nucleolin) quenching in the absence of Ire1p-RFP in these strains. (D) Histogram depicting the Relative FRET value (in arbitrary units) from donor HAC1^NRE-GFP-nucleolin mRNA into Ire1p-RFP foci at 10 minutes after ER stress by 1 μg/ml tunicamycin in a normal and cbc1-Δ strain. Donor (HAC1^NRE-GFP-nucleolin mRNA) quenching as an indicator of relative FRET value was measured for HAC1^NRE-GFP-nucleolin recruitment into Ire1p foci. Means and standard error of the mean were determined from n = 5 (biological replicates). Donor quenching was determined by Flow Cytometry from 50 000 cells of each strain were determined as described in the method section using BD Accuri™ C6 software. The statistical significance of difference as reflected in the ranges of P-values estimated from Student's two-tailed t-tests for a given pair of test strains for each message are presented with following symbols, ∗P < 0.05, ∗∗P < 0.005 and ∗∗∗P < 0.001, NS, not significant.