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. 2015 Nov 5;6(11):e1960. doi: 10.1038/cddis.2015.267

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Copyright © 2015 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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The accumulation of NLC1-C in the nucleus represses miR-320a and miR-383 transcript by binding to Nucleolin and is associated with male infertility. (a) NLC1-C accumulated in the nucleus of spermatogonia and primary spermatocytes in the testes of infertile men with mixed patterns of MA compared with normal controls. RNA in situ hybridisation was performed with DIG-labelled NLC1-C in the testes of infertile men with mixed patterns of MA and normal controls. Top: Representative images are shown for × 40 magnification. Scale bars 20 μm. The images in the red box and red arrow heads represent the nuclear signal of NLC1-C in the nucleus of spermatogonia and primary spermatocytes. All nuclei were counterstained with DAPI. (b and c) The primary and mature miR-320a expression was down-regulated in MA patients. Quantitative real-time PCR analysis of the primary and mature miR-320a expression in normal testes and MA patients testes. (d and e) NT2 cells were transfected with indicated plasmids, the accumulation of NLC1-C in the nucleus was increased and the signal of NLC1-C binding to Nucleolin was enhanced. NT2 cells were transfected individually with the indicated plasmids. Fractionated nuclear and cytoplasmic RNAs were collected 48 h after transfection and then subjected to quantitative real-time PCR assay. Transcripts of NLC1-C were probed with DIG-labelled NLC1-C combined with immunofluorescence detection of Nucleolin in NT2 cells. (f and g) The expression of primary and mature miR-320a/383 was inhibited more significantly when NT2 cells were transfected with pZW1-sno-NLC1-C plasmids compared with pcDNA3.1-NLC1-C. NT2 cells were transfected individually with the indicated plasmids for 48 h, and then subjected to quantitative real-time PCR assay. Data are presented as means±S.E.M. for at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001, compared with negative controls. (h and i) The expression of primary and mature miR-320a/383 was inhibited more significantly when NT2 cells were transfected with pZW1-sno-NLC1-C plasmids compared with pcDNA3.1-NLC1-C. NT2 cells were transfected individually with the indicated plasmids for 48 h, and then subjected to northern blotting assay. (j and k) The expression of miR-383 targets IRF1 and Cyclin D1 (j) and miR-320a targets ARPP-19 and ERRγ (k) was promoted by pZW1-sno-NLC1-C. Western blot analysis of the expression of IRF1, Cyclin D1, ARPP-19 and ERRγ after NT2 cells were transfected individually with the indicated plasmids for 48 h