Fig. 8

Inhibition of Notch1 signaling reverses the increase in WISP-1 production and the inhibition of migration, invasion and signaling pathways in vivo. The experimental design was as described in Fig. 7. a–c Representative confocal images of primary tumor sections stained with an anti-α-SMA antibody (red; a), an anti-NICD1 antibody (red; b), and an anti-WISP-1 antibody (red; c), an anti-Thy1 antibody (green), and DAPI (blue) (left). The ROI − Merge panels show high-magnification images of regions of interest as indicated by the white squares in the Merge panels. Original magnification: ×200. Scale bars = 20 μm. Quantification of α-SMA staining in Thy1⁺ cells and of NICD1⁺ cells and WISP-1⁺ cells among Thy1⁺ cells (right). The data are presented as the means ± standard errors from three mice per group (a–c right). d, e Representative confocal images of isolated Thy1⁺ CAFs from primary tumors stained with antibodies specific for α-SMA (red), NICD1 (red) or WISP-1 (green) (left). The imaging medium was VECTASHIELD fluorescent mounting medium containing DAPI. Images were acquired at ×200 magnification. Scale bars = 200 μm. Relative fluorescence intensity of α-SMA staining and quantification of NICD1⁺ cells and WISP-1⁺ cells (right). f ELISA of WISP-1 in the culture media of isolated Thy1⁺ CAFs, CD326⁺ tumor cells, and CD11b⁺ TAMs and in serum. Phase contrast microscopy of migrated and invaded CD326⁺ tumor cells (g) and Thy1⁺ CAFs (h). Scale bars: 100 µm. i Immunoblot analysis of proteins in isolated CD326⁺ tumor cells. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal‒Wallis test with Dunn’s post hoc test. The data are from three replicates per condition, with cells pooled from three mice per replicate. The data are from one experiment representative of three independent experiments with similar results (d and e left, g–i) and are shown as the mean ± standard error from three independent experiments (d and e right, f)