Figure 6

SPARCL1-activated osteosarcoma WNT/β-catenin signaling promotes macrophages recruitment by increasing CCL5 production. (a) Venn diagram depicting overlapping chemokines in three lists. List 1 included nine chemokines that have the ability to recruit macrophages. List 2 revealed 19 chemokines that have a positive correlation with SPARCL1 after analyzing in the OS database (http://hgserver1.amc.nl). List 3 contained five chemokines that might be regulated by the WNT/β-catenin pathway, as reported in the literatures. (b) Kaplan–Meier analysis of metastasis-free survival rate was related to the expression of CCL5 expression in 88 OS cases based on a human osteosarcoma gene expression database (http://hgserver1.amc.nl). (c) Expression of CCL5 mRNA in established SPARCL1-overexpression cell lines (MNNG-HOS and U-2OS) and control cells (n=3). The results shown are mean±s.d., *P<0.05. (d) Amount of secreted CCL5 in 48-h serum-free SPARCL1-overexpression compared control OS cell supernatants, assessed by ELISA (CM, conditioned medium) (n=3). The results shown are mean±s.d., *P<0.05. (e) A ChIP assay in U-2OS and MNNG-HOS cell lines was performed to confirm the potential TCF4-binding site in the CCL5 promoter region. Integration maps of chip assay are shown. IgG and input fractions were used as controls (M, maker; P1, primer 1; P3, primer 3; P9, primer 9). (f) Luciferase activities of OS cells (U-2OS and MNNG-HOS) in luciferase reporter plasmid containing wild type and mutant CCL5 promoter (mutation site: red) (n=3). The data shown are mean±s.d., ***P<0.001. (g) Luciferase reporter gene assay of CCL5 in OS cells (U-2OS and MNNG-HOS) treated with 200 ng/ml rWnt3a or vehicle (PBS containing 0.1 % bovine serum albumin) (n=3). The results shown are the mean±s.d. of relative firefly/Renilla ratio, **P<0.01, ***P<0.001. (h) Chemotaxis assay was performed using the transwell system. As shown in left panel, primed THP-1 cells (macrophages) were seeded in the upper chamber and CM from SPARCL1-overexpressing or control cells (U-2OS and MNNG-HOS) in the lower chamber. In order to explore the chemotaxis effect of CCL5, its neutralizing antibodies were added to the CM from SPARCL1-overexpressing cells (n=3). As shown in right panel, the migrated cells were counted. Representative images of migrated cells are shown in Supplementary Figure 6h. The data shown are mean±s.d., *P<0.05, **P<0.01.