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. 2020 Nov 2;11:5513. doi: 10.1038/s41467-020-19349-w

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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. 4 — a Western blotting showing the levels of Cyclin D1, phosphorylated Cyclin D1 (p-D1) (Thr286), p-D1 (Ser-90), and GSK3β in MCF7-Pa and MCF-Re cells. b–d Western blotting showing the levels of Cyclin D1, p-D1(Thr286), and GSK3β in DILA1-silenced MCF-Re cells (b), in DILA1-overexpressed MCF7-Pa cells (c), and in DILA1 and constitutively activated GSK3β (GSK3β-CA) both overexpressing MCF7-Pa cells (d). GAPDH was used as a loading control. e–g Western blotting showing the levels of nuclear and cytoplasmic Cyclin D1 in MCF7-Pa and MCF-Re cells (e), in DILA1-silenced MCF-Re cells (f), and in DILA1-overexpressed MCF7-Pa cells (g). GAPDH and Lamin A/C was used as cytoplasmic and nuclear controls, respectively. h RNA pull-down showing the interaction between Cyclin D1 and full-length or serial truncation mutants of DILA1. i eCLIP-qPCR assay indicating the exact DILA1 region responsible for Cyclin D1 binding in MCF7-Re cells (bottom). A schematic diagram of DILA1 primers (P1–P11) designed for eCLIP-qPCR, covering the full length of DILA1 (top). j RNA pull-down showing the interaction between Cyclin D1 and DILA1 or DILA mutant with deletion of hairpin A (DILA1-mA). k RNA pull-down showing the interaction between DILA1 and HA-tagged full length or truncation mutants (FL (1–295), T1 (20–295), T2 (91–295), and T3 (1–256)) of Cyclin D1 proteins in MCF-7 cells. AS (antisense) was used as a negative control. l In vitro kinase assay showing the phosphorylation of Cyclin D1 at Thr286 by active GSK3β-CA or inactive GSK3β-KD in the absence or presence of DILA1 or DILA1-mA. m RIP-qPCR of DILA1 immunoprecipitated with anti-HA antibody or IgG in MCF7-Re cells with ectopically expressed HA-Cyclin D1 or HA-Cyclin D1(T286A). n eCLIP-qPCR assay in MCF7-Re cells with HA-Cyclin D1 or HA-Cyclin D1(T286A) overexpressed. For i, m, n, n = 3 biologically independent experiments, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. o Western blotting showing the levels of Cyclin D1 and p-D1(Thr286) in MCF7-Re cells transfected with control vector or vector expressing Cyclin D1(T286A) and then transfected with NC or DILA1-ASOs. For a–h, j–l, o, representative images of three biologically independent experiments are shown.