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. 2017 Jan 11;45(8):4810–4824. doi: 10.1093/nar/gkw1345

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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 4. — (A) Schematic cartoon of the mRNA reporters whose translation is driven by the AEV or SVV IRESes and which contain four let-7 sites in either sense (blue arrows) or antisense (red arrows) orientation. Please note that the constructs harbor a 100 nucleotides poly(A) tail after in vitro transcription. (B) Capped/polyadenylated (+/+) and uncapped/polyadenylated (-/+) luciferase mRNAs driven by the β-globin (Globin +/+), the AEV (AEV -/+) or the SVV (SVV -/+) IRESes respectively were transfected together with a control Firefly luciferase mRNA in HeLa cells by lipofection (see Materials and Methods). Luciferase assays were performed after 150 min. Left panel shows the relative Luciferase expression level (ratio RLuc/FLuc) for the constructs that bear the let-7 target sites in the antisense orientation. Right panel shows the miRNA effect that is given by the difference of expression from constructs containing the let-7 sites in the antisense orientation (black bars) with those in the sense orientation (grey bars). Results are expressed as a percentage of the control (construct bearing the let-7 sites in the antisense orientation (black bars), set to 100%). (C) The same constructs as above were electroporated in HeLa cells for 1 h before reading of the luciferase activities. Left panel shows the relative Luciferase expression level (ratio RLuc/FLuc) for the constructs that bear the let-7 target sites in the antisense orientation. Middle panel shows the miRNA effect given by the difference of expression from constructs containing the let-7 sites in the antisense orientation (black bars) with those in the sense orientation (grey bars). Results are expressed as a percentage of the control (construct bearing the let-7 sites in the antisense orientation (black bars), set to 100%). Right panel shows the quantification of cytoplasmic Renilla luciferase transcripts by quantitative RT-PCR. Results are normalized to an internal mRNA (GAPDH) and are expressed as a percentage of the control (construct containing the let-7 sites in the antisense orientation, (black bars)). (D) Uncapped/polyadenylated (AEV -/+) and uncapped/non-polyadenylated (AEV-/-) constructs driven by the AEV IRES were lipofected together with a Firefly Luciferase mRNA in HeLa cells (see Materials and Methods). Luciferase assays were performed after 150 min. Left panel shows the relative Luciferase expression level (ratio RLuc/FLuc) for the constructs that bear the let-7 target sites in the antisense orientation. Right panel shows the miRNA effect which is given by the difference of expression from constructs containing the let-7 sites in the antisense orientation (black bars) with those in the sense orientation (grey bars). Results are expressed as a percentage of the control (construct bearing the let-7 sites in the antisense orientation (black bars), set to 100%). Error bars correspond to SD obtained from, at least, three different experiments. * corresponds to P-value < 0.05 obtained with a non-parametric Wilcoxon–Mann–Withney test.