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. 2020 Aug 24;19:128. doi: 10.1186/s12943-020-01246-x

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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. 6 — circSMARCA5 can form an R-loop with its parent gene. a Secondary structure prediction for circSMARCA5 using the Mfold program. The sequence KEY shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red. b The thiophosphorus nucleic acid analog ANT complementary to KEY and its mutant ANT-mut were synthesized in vitro. c Dot-blot verifying the interaction between circSMARCA and ANT or ANT-mut. d DRIP-qPCR analysis on 15-16 exon or 13-14 exon sequences of SMARCA5 to detect the association of circSMARCA5 in MCF-7 cells overexpressing ANT or ANT-mut, RNase H-treated and/or DRIP-qPCR analysis of the 15-16 exon sequence as a control. “*” indicates P < 0.05. e Western blot analysis shows that transfection of ANT into circSMARCA5-overexpressing cells can restore SMARCA5 protein levels but ANT-mut cannot. (f, g) Dot-blot analysis quantifying R-loop strength between the SMARCA5 locus and circSMARCA5 or circSMARCA5-mut (guanine converted to cytosine of the KEY sequence). h DRIP-qPCR in MCF-7 cells transfected with circSMARCA5 or circSMARCA5-mut. RNase H-treated genomic DNA and qPCR of exon13-14 were treated as controls. “*” indicates P < 0.05. i Western blot analysis shows that overexpression of circSMARCA5 to MCF-7 cells can decrease SMARCA5 protein levels but circSMARCA5-mut cannot