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. 2020 Nov 13;3:677. doi: 10.1038/s42003-020-01404-3

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© The Author(s) 2020

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. 2 — a Venn diagram of miRNAs whose expression is associated with a good prognosis in ovarian cancer patients (MiROvaR signature³³) showing the overlap (n = 3) between miRNA predicted to target the ET_AR 3′UTR (n = 5, red circle) and miRNA predicted to target the ZEB1 3′UTR (n = 7, green circle). b Expression levels of ET_AR and miR-200b or miR-200c in the indicated cell lines. The ratio of ET_AR/β-actin, evaluated by WB and miR-200b/c expression, evaluated by qRT-PCR and normalized to U6, are shown as bar and line, respectively. Values are the mean ± SD (n = 3). c ET_AR mRNA levels in HEY and SKOV3 cells transfected for 48 h with mimic-miRNA control (miR-Ctr), mimic-miR-200b (miR-200b) or mimic-miR-200c (miR-200c) evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD (n = 3; *p < 0.001 vs. Ctr). d Lysates from HEY and SKOV3 cells transfected as in c are analyzed by WB for ET_AR expression. β-actin is used as loading control. e Luciferase activity in HEY cells co-transfected for 48 h with miR-Ctr, miR-200b, or miR-200c together with the reporter plasmid containing the 3’UTR region of ET_AR (ET_AR 3′UTR) or its triple mutant (mut/ΔET_AR 3′UTR). Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.001 vs. Ctr). f WB for ET_AR expression in the lysates from HEY cells 48 h transfected with miRNA control, miR-200b or miR-200c inhibitors (anti-miR-Ctr, anti-miR-200b, and anti-miR-200c). β-actin is used as loading control. g Cell viability of HEY cells, transfected as in c and f, or with siRNA control (si-Ctr), or si-ET_AR, and treated or not with macitentan (MAC) for 48 h. Values are the mean ± SD (n = 4; *p < 0.001 vs the respective Ctr). h Lysates of HEY cells transfected as in c together with a plasmid control (Mock), a wt ET_AR expression plasmid, or with an ET_AR expression plasmid w/o the 3′UTR as indicated are analyzed by WB for ET_AR expression. β-actin is used as loading control. i Cell vitality of HEY cells transfected as in h. Values are the mean ± SD expressed as fold induction (n = 4; *p < 0.001 vs. Mock; **p < 0.001 vs. wt ET_AR).