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. 2017 Dec 13;8:2103. doi: 10.1038/s41467-017-02282-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — GAr-induced mRNA translation stress stimulates cell proliferation via c-Myc. a Colony forming assay. The number of colonies of H1299 cells expressing the gly-ala repeat (GAr) of the EBV-encoded EBNA1, or plasmid control (EV), were counted after 15 days in selection medium and plotted. b The graph shows the percentage of proliferating cells (S-phase) expressing the GAr or EV under indicated growth conditions as determined by FACS analysis. c Western blots (lower panel) show that fusing the GAr to the N terminus of the open reading frames of chicken ovalbumin (Ova), (GAr-Ova), p53 (GAr-p53) or GFP (GAr-GFP) suppresses mRNA translation, in contrast to when inserted at the C-terminal end (Ova-GAr or p53-GAr). The graph (upper panel) shows the relative number of cells in S-phase expressing the indicated constructs. d Western blots show the expression of c-Myc following expression of the indicated quantities of GAr cDNA in H1299 cells. The numbers below the blots indicate c-Myc levels normalised to actin. e Fusing the 5′ UTR of c-myc that contains IRES to the 5′ of GAr-Ova (IRES GAr-Ova) abrogates translation suppression and averts c-Myc induction. f The upper graph shows RT-qPCR of c-myc mRNA levels at indicated time points following expression of the GAr. The data show the relative c-myc/GAPDH mRNAs ratio and the highest value at 36 h is set to 100%. The lower graph shows the relative values of c-Myc-induced CAD mRNA levels relative to GAPDH. The highest value is set to 100%. g RT-qPCR analysis show the relative c-myc mRNA levels normalised to GAPDH in H1299 cells expressing EV, EBNA1 WT or EBNA1 ∆GAr. h RT-qPCR analysis of c-myc target genes (Cdk4, cyclin d1, 45S pre-rRNA, Rps19 and Rpl38) in cells expressing EV or GAr. Note the logarithmic scale. FACS analyses show mean values with s.d. from at least three independent experiments. Actin was used as a loading control. For western blots, RT-qPCR and colony formation assays representative of three independent experiments were shown with s.d. Statistical significance was calculated using t tests (***p < 0.001, **p < 0.05 and *p < 0.1)