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. 2024 Feb 8;25(3):1022–1054. doi: 10.1038/s44319-024-00075-z

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© The Author(s) 2024

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 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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV4 — (A) Schematic representation of the TCF/LEF-luciferase reporter assay. Activators (Wnt ligands, CHIR) and inhibitors (XAV939) of TCF/LEF-dependent responses are also shown. (B) TCF/LEF-luciferase reporter assay in HuCCT1 cells transiently transfected with TOP-Flash or FOP-Flash luciferase expression vectors and treated with CHIR99021 or DMSO for 24 h. Cells were co-transfected with a Renilla luciferase expression vector for normalization of the firefly luciferase activity. (C) TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with CHIR99021 or DMSO for 24 h. TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with the indicated concentrations of TGFβ1 or BSA/HCl (Ctr) for 16 h. (D) Subcellular fractionation followed by immunoblotting for β-catenin, lamin B1 (nuclear marker), β-tubulin and 14-3-3 (cytoplasmic markers) using nuclear and cytoplasmic fractions of HuCCT1 cells stably over-expressing an empty vector (pcDNA3.1) or LINC00313 and treated with CHIR99021 or DMSO for 24 h. (E) Immunofluorescence to detect β-catenin subcellular localization in control or LINC00313 over-expressing HuCCT1 cells, treated or not with CHIR99021 or DMSO for 24 h. Scale bars: 150 μm. (F) Cell viability assays in control or LINC00313 over-expressing HuCCT1 cells treated with CHIR99021 or DMSO for 24 h. (G) Colony formation assay in control or LINC00313 over-expressing HuCCT1 cells, treated with CHIR99021 or XAV939 or DMSO for 24 h. Quantification of the number of colonies for each condition is also shown. (H) Real-time qPCR analysis of AXIN2, TCF7 and LINC00313 expression in control or LINC00313 overexpressing HuCCT1 cells, treated with CHIR99021, XAV939, or DMSO for 24 h. Data information: In panel (B) data are presented as mean ± SD (n = 3 biological replicates). In panel (F) data are presented as mean ± SD (n = 6 biological replicates). **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test). In panels (G) and (H) data are presented as mean ± SD (n = 3 biological replicates). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test). Source data are available online for this figure.