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. 2021 Mar 4;40(13):2407–2421. doi: 10.1038/s41388-021-01718-4

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© The Author(s) 2021, corrected publication 2021

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 LNCaP or PC3 cells were knocked down by control siRNA (siNC) or siRNA against COUP-TFII (siCII) for 48 h. Western blot results showed the knockdown efficiency of siRNA against COUP-TFII (upper panel). miR-21 and RNU6B expression levels were measured by realtime PCR (n = 3) and results were presented as miR-21 normalized to RNU6B (lower panel). Asterisk indicates p < 0.05 using two-tailed Student’s t-test. B Doxycycline (0.5 μg/ml) was used to treat RWPE-1 cells carrying with empty vector (vector) or inducible COUP-TFII construct (i-CII) for 48 h. Western blot result demonstrated the expression level of COUP-TFII after doxycycline treatment (upper panel). miR-21 and RNU6B expression levels were measured by realtime PCR (n = 3) and results were presented as miR-21 normalized to RNU6B (lower panel). Asterisk indicates p < 0.05 using two-tailed Student’s t-test. C LNCaP cells were used to perform COUP-TFII Chromatin- immunoprecipitation (ChIP)-PCR. Cartoon of miR-21 locus showed the two potential COUP-TFII binding regions. Specific primers were individually designed to amplify those regions. Results were presented as percentage of input control (n = 3). Asterisk indicates p < 0.05 using two-tailed Student’s t-test. D Expression levels of miR-21 and LATS1 were measured in parental LNCaP (WT) cells and enzalutamide-resistant (enzaR) clones by realtime PCR (n = 3). Asterisk indicates p < 0.05 using one-way ANOVA test following by Dunnett analysis. E EnzaR cells were treated miR-21 inhibitor (miR-21-i) for 48 h and then performed Ago2-RNA-immunoprecipitation (RIP). Specific primer was designed to amplify LATS1 3′UTR region containing miR-21-targeting site. Results were presented as percentage of input control. Asterisk indicates p < 0.05 using two-tailed Student’s t-test. F EnzaR cells were co-treated control mimic or miR-21 mimic (50 nM) and control siRNA or siRNA against COUP-TFII (40 nM) for 48 h. COUP-TFII (CII), YAP1, LATS1, and β-actin expression levels were detected by western blot.