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. 2018 Jul 23;9(8):803. doi: 10.1038/s41419-018-0821-5

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© The Author(s) 2018

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. 3 — a Putative miR-181 family binding site in lincRNA-p21. The potential complementary residues are underlined and mutated bases are indicated in red. b Upper panel, schematic representation of pmirGLO-based luciferase reporter plasmid containing wild-type lincRNA-p21 (Luc-lincRNA-p21-wt) and a mutant reporter construct (Luc-lincRNA-p21-mut). Lower panel, relative luciferase activity of the luciferase constructs of Luc-lincRNA-p21-wt or Luc-lincRNA-p21-mut co-transfected with miR-control/miR-181 family in HEK293 cells. A representative experiment with triplicates of two independent experiments is shown. Data are expressed as mean ± SEM of triplicate wells. *P < 0.05, **P < 0.01 in one-way analysis of variance ANOVA followed by Bonferroni test. c, d qRT-PCR analysis of lincRNA-p21 in BV2 microglia cells treated with LPS following transfection of cells with miR-control/miR-181a, b, c, d mimics (c); or anti-miR-control/anti-miR-181 a, b, c, d (inhibitors) (d). Data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control using one-way analysis of variance ANOVA followed by Bonferroni test. e, f Anti-Ago2 RIP was performed in BV2 microglia cell lysates. Co-precipitated RNAs were used to detect the relative enrichment of lincRNA-p21 (e) and miR-181 family (f) associated with Ago2, compared with that in normal IgG control. g, h In vitro-synthesized biotin-labeled RNA pulldown experiments in BV2 microglia cells. BV2 microglia cell lysates were incubated with in vitro-synthesized biotin-labeled lincRNA-p21 sense or antisense RNA for biotin pull-down assay, followed by qRT-PCR to detect miR-181 family (g) and western blotting to detect Ago2 (h) associated with lincRNA-p21. Bio-linc-AS, biotinylated-lincRNA-p21-antisense RNA; bio-lincRNA-p21, biotinylated-lincRNA-p21-sense RNA. i Representative analysis of lincRNA-p21 and miR-181 family distribution in nuclear and cytoplasmic fractions in BV2 microglia cells. U2 snRNA and 18S rRNA served as controls for nuclear and cytoplasmic RNA, respectively. Exogenous spike-in of cel-mir-39 served as a normalizer for the qRT-PCR analysis. j, k Biotin pull-down assay with biotin-labeled lincRNA-p21 sense or antisense RNA was performed with the cellular fractions of cytoplasm (j) or nucleus (k) of BV2 microglia cells. A representative experiment with triplicates of two independent experiments is shown. Data are expressed as mean ± SEM of triplicate wells. *P < 0.05, **P < 0.01 ***P < 0.001 in two-way analysis of variance ANOVA followed by Bonferroni test