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. 2018 Jul 24;37(48):6259–6274. doi: 10.1038/s41388-018-0409-7

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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. 6 — Enhanced invasion capacity mediated by ectopic T:E fusion or ERG in AR-negative prostatic cells is under ERRα regulation. a Schematic diagram shows the construction of the lentiviral plasmid expressing the T:E fusion or ERG under the regulation of ERRα. An approximate 2-kb fragment of the innate T:E fusion gene promoter including the P1 ERRα-binding element and the full-length open reading frame (ORF) of the T:E fusion gene was cloned from the genomic DNA and cDNA respectively from the T:E fusion-positive VCaP cells. The two DNA fragments were PCR-ligated and inserted into the modified CMV promoter-deleted lentiviral expression vector pLenti6-V5-TOPO as pLenti6-P-T:E. Under the regulation of its innate promoter containing the P1 ERRα-binding element, the generated pLenti6-P-T:E expresses the T:E fusion or ERG. b and c Immunoblot analyses of ERRα and ERG in T:E fusion stable infectants of PC-3 and BPH-1 cells. Results validated that ERG was expressed in both PC-3-T:E and BPH-1-T:E infectants, with no alteration upon dihydrotestosterone (DHT) treatment. d and e RT-qPCR and immunoblot analyses of ERRα and ERG in ERRα-knockdown T:E infectants. Results showed that both mRNA and protein levels of ERG were significantly reduced in both PC-3-T:E and BPH-1-T:E infectants upon shRNA-induced knockdown of ERRα. f and g Transwell invasion assay on PC-3-T:E and BPH-1-T:E infectants. Upper panels: representative images show the crystal violet-stained invading T:E infectants, magnification 100×. Analysis showed that both PC-3-T:E and BPH-1-T:E infectants exhibited significant higher invasion capacity than their control counterpart vector-infectants. The invasion capacities of both T:E infectants were significantly suppressed upon shRNA-induced knockdown of ERRα. Figures above the images indicate the relative expression levels of ERG in respective clones. ^*P < 0.05; ^**P < 0.01 versus empty vector-infectants, two-tailed Student’s t test. All data are presented as mean ± SEM obtained from three independent experiments