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. 2020 Mar 26;13:24. doi: 10.1186/s13045-020-00857-7

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© The Author(s). 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — HNF4A-AS1 facilitates growth and invasion of NB cells via hnRNPU-mediated transactivation of CTCF. a Volcano plots of RNA-seq revealing alteration of gene expression (fold change > 2.0, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1. b By analyzing with ChIP-X program and BioGRID database, venn diagram (left panel) showing identification of hnRNPU-interacting transcription factors (TFs) regulating genes altered in RNA-seq and correlated with HNF4A-AS1 in a public dataset (GSE45547). Gene network (right panel) revealing identified TFs and target genes. c Co-IP and Western blot assays indicating the interaction between hnRNPU and CTCF in SH-SY5Y and BE(2)-C cells stably transfected with mock, HNF4A-AS1, scramble shRNA (sh-Scb), or sh-HNF4A-AS1 #1. d Confocal images of BiFC assay showing direct interaction between hnRNPU and CTCF (arrowheads) within SH-SY5Y and BE(2)-C cells co-transfected with pBiFC-VN173-hnRNPU and pBiFC-VC155-CTCF, and those stably transfected with mock, HNF4A-AS1, sh-Scb, or sh-HNF4A-AS1 #1. Scale bars, 10 μm. e ChIP and real-time qPCR (normalized to input) assays indicating the CTCF enrichment on target gene promoters in SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-hnRNPU (n = 5). f and g Real-time qRT-PCR (f, normalized to β-actin, n = 4) and Western blot (g) assays showing the levels of HNF4A-AS1, hnRNPU, CTCF, and target genes in SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-Scb, sh-hnRNPU #1, or sh-CTCF #3. h and i Representative images (left panel) and quantification (right panel) of soft agar (h) and matrigel invasion (i) assays indicating anchorage-independent growth and invasion of SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-hnRNPU #1, sh-CTCF #3, or sh-HNF4A #2 (n = 5). ANOVA compared the difference in e, f, h, and i. *P < 0.05 vs. mock+sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c–i