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. 2019 Jan 10;10:130. doi: 10.1038/s41467-018-07987-0

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© The Author(s) 2019

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. 5 — DKK3 potentiates β-catenin and YAP/TAZ signalling in CAFs. a Graph showing GSEA of murine CAF1 after transfection with control siRNA (CAF-siCtr) vs CAF1 after transfection with DKK3 siRNAs (si#2 and si#3). The graph indicates the Normalised Enrichment Score (NES) and False Discovery Rate (FDR) q-value (-log₂) for each gene set. In green, top differential gene-sets, including β-catenin and YAP/TAZ. b Western blot showing DKK3, non-phospho (active) β-catenin (Ser33/37/Thr41), β-catenin, TAZ,YAP and Gapdh levels in CAF1 after transfection with control (siCtr) and two independent DKK3 siRNA. c Images show YAP (left panels) or TAZ (right panels) localisation (green) and DAPI staining (red) in CAF1 after transfection with control (siCtr) and two independent DKK3 siRNA. Scale bars, 20 µm. d Tukey boxplots show luciferase activity (Firefly/Renilla) indicative of β-catenin activation (TOPFlash reporter) or YAP/TAZ activation (4xGTIIC-lux reporter) in CAF1 after transfection with control (siCtr) or DKK3 (si#2&3) siRNAs; n = 9 for β-catenin, n = 17 for YAP/TAZ. e Left panels show total β-catenin (green), S100A4 (CAF marker, red) and DAPI (blue) staining of TS1 tumours admixed with WT or KO.9-CAFs. Right panels show processed images showing β-catenin staining in CAF-positive areas. Scale bar, 100 µm. Chart shows mean β-catenin intensity in CAFs. Bars represent mean ± SEM (n = 4 fields of view). f Left panels show total YAP (green), S100A4 (CAF marker, red) and DAPI (blue) staining of TS1 tumours admixed with WT or KO.9-CAFs. Right panels show processed images showing YAP staining in CAFs. Scale bar, 100 µm. Chart shows mean YAP intensity in CAFs. Bars represent mean ± SEM (n = 19 or more fields of view). g Graphs show correlations between DKK3 gene expression and the expression of CAF-specific YAP/TAZ and β-catenin signatures (z-score normalised) in normal and cancerous stroma from mammary gland, colorectal and ovarian human tissues. Pearson correlation coefficient (r) and p values are shown. h Representative Western blot showing non-phospho (active) β-catenin (Ser33/37/Thr41), β-catenin, TAZ, phospho-S127-YAP, YAP and tubulin in murine NF1, N4, CAF1 and CAF5. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001; n.s., non-significant