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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 activity and levels are highest when cells are in G2/M. (A,B) miRNA activity in untreated HER10 cells segregated by cell cycle phase using flow cytometry. (A) Representative examples of flow cytometry data quantifying the number of GFP+ cells in let-7s-transfected (top panels) or miR-1s-transfected (bottom panels) HER10 cells. The percentage of GFP+ cells in let-7s transfected samples is higher in G1 (black box, upper left panel) as compared to S and G2/M (black boxes, upper middle and right panels). However, the percentage of GFP+ cells in miR-1s transfected samples is similar in all phases of the cell cycle (bottom panels). (B) Quantification of flow cytometry data shown in (A). N = 3 experiments; 40,000 events per experiment. p = 0.0063 and p = 0.0055 for Let-7s S and G2M (v.s. G1) respectively. (C) qRT-PCR analysis of let-7a,b,c,d,f in G1, S, and G2/M phase populations segregated using FACS with ClickIt-EdU and DAPI. With the exception of Let-7b, which is highest in S-phase, all let-7 family members are highest in the G2/M population. N = 3 experiments; with > 500k cells per population. p = 0.012 (let-7a G2M), p = 0.023 (let-7b S), p = 0.039 (let-7c G2M), p = 0.019 (let-7d G2M), and p = 0.039 (let-7f G2M).