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. 2019 Oct 25;9:15336. doi: 10.1038/s41598-019-51703-x

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© The Author(s) 2019

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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Let-7 is active in human embryonic retina-derived HER10 cells. (A) Schematic of a miRNA sensor. miRNA target sites are added to the 3’UTR end of GFP. If the miRNA of interest is present, GFP will be repressed (middle panel in A). In contrast, when no miRNA is present (left panel) or when a miRNA antagomiR is added (right panel), GFP is abundantly expressed. (B) GFP is present in 20–30−1.64 of HER10 cells transfected with pCAG-mCherry (red) and pLVX-let-7 sensor (let-7s). (C) Sensor-GFP expression is repressed when let-7 levels are increased via transfection of pLV-let-7 (OE; compare B’ to C’). (D) Co-transfection with a let-7 antagomiR (Ant) leads to GFP expression in almost all transfected cells. (E) Nearly all cells transfected with a miR-1 sensor (miR-1s) express GFP (E’). (F) Western blot analysis of a cyclohexamide time-course (time (h) = time after cyclohexamide addition) to determine the half-life of AcGFP1 present in the let-7s construct. The half-life of AcGFP1 is short, approximately 3.5 h (left and bottom panels), as compared to eGFP which was > 24 h (right panel). Unaltered images of Western Blots are included in the Supplementary Information (See Fig. S4).