Fig. 8.

DKK3 regulates the balance between LRP6 and Kremen and affects YAP/TAZ signalling. a Western blots show anti-Flag co-immune-precipitation of LRP6 and Kremen1 in human BC-CAFs (TB147) expressing Flag-DKK3 or empty vector (null). b Images show DKK3 (green), Kremen1 (red) and DAPI (blue) staining of human BC-CAF TB165 after transfection with control (siCtr) and DKK3 siRNA (smart-pool). Scale bars, 15 µm. c Images show LRP6 (green), Kremen1 (red) and DAPI (blue) staining of human BC-CAF TB165 transfected as in (b). Scale bars, 15 µm. d Western blots show LRP6, Kremen1, Kremen2, DKK3 and Gapdh expression in WT and KO.9 CAFs. e Western blots show LRP6, Kremen1 and tubulin expression in indicated CAFs after treatment with 100 μg mL⁻¹ cyclohexamide (CHX) at different times (in min). Graph represents quantification of the indicated blots (normalised to tubulin) at 180 min relative to the amount at 0 min. f Western blots show non-phospho (active) β-catenin (Ser33/37/Thr41), TAZ, Kremen1 and Tubulin CAF-KO.9 after transfection with control (siCtr) and Kremen1&2 siRNA (smart-pool). g Graph shows gel contraction of KO.9 CAFs after transfection with control (siCtr), Kremen1 (siKr1), Kremen2 (siKr2) or Kremen1&2 (siKr1&2) siRNAs (smart-pool). Bars represent mean ± SEM (n = 4 or more individual gels). h Graph shows the invasion index of TS1 spheroids after 3D co-culture with KO.9 CAFs transfected as in (f). Bars represent mean ± SEM (n = 11 or more individual spheroids, 2 independent experiments). i Graph shows the tumoral area of TS1 MOT co-culture with KO.9 CAFs transfected as in (f). Bars represent mean ± SEM (n = 348 or more colonies, at least 3 independent experiments). j Images show LRP6 (green), GM1 (red) and DAPI (blue) staining of indicated CAFs. Cells were fixed and subjected to staining without permeabilisation. Scale bar, 15 μm. k Images show YAP (green) and DAPI (red) staining of KO.9 CAFs transfected as in (f). Bar, 30 µm. Graph shows quantification of nuclear relative to cytosolic fluorescent intensity (log₁₀ ratios) of YAP. Lines represent mean ± SEM. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001; n.s., non-significant