Extended Data Fig. 2: Validation of E0771 primary tumor from Net4 knockout and wildtype mice.
From: Basement membrane stiffness determines metastases formation
a, Immunofluorescence and immunohistochemistry staining of primary E0771 tumors grown in Net4 wildtype (WT) and Net4 knockout (KO) animals. Different stromal cell types were detected using αSMA (fibroblasts), F4/80 (macrophages), and CD8a (T-cells). Scale bar, 100 µm. Box and whiskers blot of cell type amount within primary tumor tissues (two-tailed Unpaired t test, Min to Max, median is shown as line, mean is displayed as cross, data points appear as grey dots; n = 6; t = 0.2948 (αSMA), t = 0.3001 (F4/80), t = 0.1246 (CD8a), df = 10; ns, not significant). b, Proliferation of cancer cell lines (E0771, 4T1, MDA-MB-231, and HCmel12) treated without (ctrl) and with different concentrations of Net4 (0.1, 3.6, 5.2 µg/well) for 96 h. Absorbance was normalized to ctrl. Scatter dot blot together with bars of proliferation assay (E0771: Kruskal-Wallis test, mean is the top of the bar; STDEV; n = 12 (ctrl and 0.1 µg/well), n = 9 (3.6 and 5.2 µg/well); ns, not significant; **P < 0.01, ***P < 0.001), (4T1, MDA-MB-231, and HCmel12: Ordinary one-way ANOVA test, mean is the top of the bar; n = 11 (4T1 and MDA-MB-231: ctrl), n = 12 (HCmel12: ctrl), n = 9 (3.6 and 5.2 µg/well); F = 5.938, DF = 37 (4T1); F = 1.254, DF = 37 (MDA-MB-231); F = 1.430, DF = 38 (HCmel12); ns, not significant; *P < 0.05, **P < 0.01). c, Contraction assay of cancer-associated fibroblast cell lines (mCAF1 and mCAF2) within collagen c1 and 7.2 µg/well) of recombinant Net4 (Net4) and its respective laminin binding mutant Net4 E195A,R199A (Mut) for 72 h. Representative image of the contraction assay showing the contracted area at the well rim. Scatter dot blot together with bars of contraction assay, in which all values are normalized to the control (Ordinary one-way ANOVA test, mean is the top of the bar; STDEV; n = 9; ns, not significant).
