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. 2022 Sep 6;15:128. doi: 10.1186/s13045-022-01348-7

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© The Author(s) 2022

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 — circPDK1 interacts with BIN1 and enhances BIN1 ubiquitination. A RNA pull-down verified the interaction of circPDK1 with BIN1. β-actin was used as a negative control. RIP assay was used to determine the relative expression of circPDK1 with rabbit BIN1 and IgG antibodies in MIA PaCa-2 cells. B Co-localization of circPDK1 (RED) with BIN1 proteins (GREEN), respectively, in MIA PaCa cells. Scale bar = 5 μm. C The expression of BIN1 in protein level after circPDK1 overexpression or loss of circPDK1. D The BIN1 protein level in MIA PaCa-2 and PANC-1 cells with circPDK1 overexpression treated with MG132 (20 μM) for 12 h. E The BIN1 protein level in indicated time point after treated with cycloheximide (CHX, 10 µg/mL) in transfected PC cells. F The interaction of circPDK1 truncations with BIN1 was confirmed by RIP assay in 293 T cells (left) and schematic diagram of circPDK1 full-length and truncations (right). G Interaction profile between circPDK1 and BIN1 was predicted by catRAPID. H RIP assays were performed using anti-BIN1 antibodies in MIA PaCa-2 cells by RT-PCR. I RNA pull-down assay was used to determine the interaction between circPDK1-WT or circPDK1-MUT and BIN1. J BIN1 mRNA and protein level were detected after transfected with circPDK1-WT or circPDK1-MUT. K IP assays verified the ubiquitination modification level of BIN1 in MIA PaCa-2 cells with indicated treatments. L The specific binding sites between circPDK1 and BIN1 was accurately construct by Atomic Rotationally Equivariant Scorer (ARES) (top) and the protein domain of BIN1 queried from UniProt (bottom). M RNA pull-down assays was performed to verify the interaction between truncated BIN1 protein and circPDK1 (top) and schematic of truncated BIN1 protein (bottom). N The expression of BIN1 in PC tumor tissues and matched normal tissues verified by IHC assays in tissue microarrays. Scale bar = 1000 μm. O The correction of circPDK1 and BIN1 protein level was verified by IHC assays in tissue microarrays. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance