Figure 5

CXCR7 regulates M-CSF, MMPs, and VCAM-1 expression. (A) Murine macrophages (RAW 264.7) were serum starved for 4 hours and plated on the top chamber of 8-μm-pore polycarbonate membrane filters and SFM containing 4T1 or 4T1 sh-CXCR7 CM (50 to 100 μg/ml) was placed in the lower chamber. After 12 hours of incubation, cells that migrated across the filter toward SFM with or without CM (50 to 100 μg/ml) were fixed, stained, and counted by using bright-field microscopy in five random fields. (B) Conditioned media (CM) obtained from Vector or CXCR7-downregulated cells were subjected to cytokine profiling. (C) The array data were quantitated by ImageJ to generate a protein profile (histogram) (D) Murine macrophages (RAW 264.7) were serum starved for 4 hours and treated with 50 and 100 μM ki20227 (MCSF-R inhibitor) and then plated on the top chamber of 8-μm-pore polycarbonate membrane filters. SFM, in the absence or presence of 4T1 CM (100 μg/ml), was placed in the lower chamber. After 12 hours of incubation, cells that migrated were fixed, stained, and counted by using bright-field microscopy in five random fields. (E) CM obtained from vector or CXCR7-downregulated cells was subjected to gelatin zymography for MMPs activity. (F) CM obtained from 4T1.2 cells treated with CXCR7-specific (CCX771) at 0.5 and 1 μM concentration or STAT3-specific inhibitors (S31-201) at 5 and 10 μM concentration in the presence and absence of CXCL12 (100 ng/ml) were subjected to gelatin zymography. (G) 4T1 and 4T1 shCXCR7 cell line-derived tumors were lysed and analyzed for VCAM-1, MMP-9, and GAPDH expression by Immunoblotting. The 4T1 Vec, 4T1 sh-CXCR7 (H), and 4T1.2 (I) cell line-derived tumors from CCX771 or S31-201 treated or untreated groups were subjected to immunohistochemical staining for MMP-9 expression (40×). Data represent the mean ± SD per experimental group. Scale bars, 0.03 mm. ^**P < 0.01, ^***P < 0.001 versus none and ^###P < 0.01 versus control.