Figure 3

SPARCL1 activates the WNT/β-catenin pathway by promoting nuclear translocation of β-catenin. (a) The distribution of β-catenin in OS cells (U-2OS and MNNG-HOS) transfected with Lenti-vector, Lenti-SPARCL1 or treated with 10 μg/ml rSPARCL1 were analyzed by IF staining. β-catenin is shown by green fluorescence, and the cell nuclei was stained with DAPI (blue fluorescence), Scale bars, 100 μm. (b) In the upper panel, the expressions of β-catenin from whole cell lysates in control and SPARCL1-overexpression cells (U-2OS and MNNG-HOS) were detected by western blotting. Nuclear proteins of control and SPARCL1-overexpression groups (U-2OS and MNNG-HOS cells) were extracted. In the lower panel, the nuclear β-catenin expressions in each group were also detected by western blotting. (c) The distribution of β-catenin in mice lung tissues was examined by IF. Representative images of β-catenin (green) in mice lung tissues are shown, the cell nuclei was stained with DAPI (blue). Scale bars, 500 μm. (d) Spearman correlation analysis between SPARCL1 expression and number of nuclear β-catenin+ OS cells based on the IHC staining (r=0.4813, P=0.0017) (HPF, high power field). (e and f) The cell migration and invasion abilities were determined by transwell migration and invasion assay in U-2OS and MNNG-HOS cells treated with 10 μg/ml rSPARCL1, 10 μM XAV-939, si-NC, si-Fzd3, si-Fzd6 or si-Fzd8 (n=3). Representative images of migrated or invaded cells are shown in Supplementary Figure 5a. Scale bars, 200 μm, values are mean±s.d., *P<0.05, **P<0.01, ***P<0.001. (g) Real-time qPCR analyses of relative expression of indicated genes in MNNG-HOS cells and MNNG-HOS cells treated with XAV939 combined 10 μg/ml rSPARCL1 normalized to MNNG-HOS cells treated with rSPARCL1 alone (n=3). Values are mean±s.d., *P<0.05, **P<0.01, ***P<0.001.