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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. 3 — DKK3 is a crucial regulator of CAF functions. a Western blots showing DKK3 and Gapdh expression in murine CAF1 after transfection with control (siCtr) and 2 independent DKK3 siRNAs (si#2 and si#3). b Colour-coded grid showing fold expression of genes generally associated to CAFs in NF4 and wild-type CAF1 after transfection with control and 2 different DKK3 siRNAs. Colours range from red to blue representing, respectively, the lowest (zero) and highest fold activity (one). Genes are grouped into CAF markers, extracellular matrix (ECM) components/remodellers, secreted factors and transcription factors (TFs). Expression for Dkk3 is shown in a scale of green colour. c Histogram shows gel contraction by CAF1 after transfection with control (siCtr) and 2 independent DKK3 siRNAs. Bars represent mean ± SEM (n = 6). Images show representative gels remodelled. d Images show F-actin (magenta) and collagen second harmonic (SHG, green) in gels remodelled by CAF1 after transfection with control (siCtr) and 2 different DKK3 siRNA. Scale bar, 50 µm. e Histogram shows Young’s elastic modulus of NF1 or CAF1 cells following transfection with control (siCtr) and DKK3 siRNA (si#sp, smart-pool). Lines represent mean ± SEM (n = 34 or more individual measurements). f Western blot showing levels of DKK3 and tubulin in wild-type CAF1 (WT) and two sets of DKK3 knockout CAF1 clones (KO) and their recovery counterparts where DKK3 was stably re-expressed (KO-REC). g Cartoon describing the experimental set-up for the 3D co-culture system. Cancer cell spheroids are embedded in a collagen:Matrigel matrix containing fibroblasts and fed with media containing 10% FBS for 4–7 days. h Images show representative end-point TS1 murine breast cancer spheroids (red) obtained after 3D co-culture with WT, KO.9 and KO.9-REC CAFs (in blue). Spheroids obtained by mono-culture or by co-culture with NFs (in blue) are also shown. DAPI staining (green) was also used. Scale bar, 200 µm. i Tukey boxplots show the invasion index (3D invasion) and tumoral area (3D growth) measured from spheroids described in (h); n > 26 individual spheroids out of 3 independent experiments. Where indicated, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001; n.s., non-significant