Figure 2

SPARCL1 inhibits osteosarcoma metastasis in vitro and in vivo. (a) SPARCL1 suppressed osteosarcoma cells (U-2OS and MNNG-HOS) migration in vitro. In the left panel, cellular migration ability in overexpression and control groups (U-2OS and MNNG-HOS cells) were detected by transwell migration assay. In the right panel, MNNG-HOS and U-2OS cells treated with different concentrations of rSPARCL1 or PBS were also used to perform transwell migration assay (n=3). Six randomly selected fields were photographed and the numbers were counted. Representative images of cellular migration conditions in each group are shown in Supplementary Figures 1c and f. Values are means±s.d., **P<0.01, ***P<0.001. (b) SPARCL1 suppressed osteosarcoma cells (U-2OS and MNNG-HOS) invasion in vitro. Similarly, cellular invasion ability was detected by transwell invasion assay in the left panel. In the right panel, MNNG-HOS and U-2OS cells treated with different concentrations of rSPARCL1 or PBS were used to perform transwell invasion assay (n=3). Representative images of invaded cells in each group are shown in Supplementary Figures 1c and f. Values are means±s.d., **P<0.01, ***P<0.001. (c) Control and SPARCL1-overexpression MNNG-HOS (Luc) cells were injected into the tail vein of BALB/C nude mice. After 30 days, the in vivo tumor metastasis was imaged. Representative images of mice in each treatment group are shown. (d) Representative images of collected lungs in control and SPARCL1-overexpression groups are shown in the upper panel. Black arrows indicate metastases. Representative photographs of hematoxylin and eosin staining of lung tissues are also shown in the lower panel. Scale bars, 50 μm. (e) Numbers of lung metastatic foci were counted in each group. Values are means±s.d., ***P<0.001. (f) Functional classifications of the 322 DEGs based on GO analysis. Shown are pie charts of gene categories (percentage indicated: invasion/metastasis-related genes: 18%; WNT pathway-related genes: 4%; cytokine production-related genes: 5%; ossification-related genes: 3%; metabolism-related genes: 9%; embryo development-related genes: 4%; neural development-related genes: 5% and apoptosis-related genes: 4%). (g) Representative GO (GO: biological process) categories affected by SPARCL1 expression in MNNG-HOS cells. (h) Heat map of cell motility molecules (GO: 0048870) from gene expression profiling results based on GO analysis (control groups vs SPARCL1-overexpressing groups, P=3.34E−5). (i) Representative KEGG pathway categories affected by SPARCL1 expression in MNNG-HOS cells.