Figure 1

CXCL12 enhances CXCR7-mediated cell migration and signaling. (A) 4T1 Vec and CXCR7 shRNA-transfected cells were subjected to chemotaxis toward CXCL12 (50 and 100 ng/ml) by using the modified Boyden chamber assay, as described in Materials and methods. (B) 4T1 Vec and CXCR7 shRNA-transfected cells were grown to confluence in complete medium in six-well plates, and then a wound was made with a 200-μl pipette tip, and the closure of the wounds was monitored in the presence or absence of CXCL12 (50 and 100 ng/ml) by microscopy after 24 and 36 hours. (C) Quantitative analysis of percentage of wound closure. (D) 4T1 and (E) 4T1.2 cells were pretreated for 1 hour with vehicle or CCX771 (1 μM) and were subjected to chemotactic assay in the absence or presence of CXCL12 (100 ng/ml). (F) 4T1 Vec and 4T1 sh-CXCR7 cells were serum starved for 4 hours and stimulated with CXCL12 (100 ng/ml) for different times, as indicated, and incubated at 37°C. After treatment, cells were washed, lysed, and analyzed with Western blotting for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), and GAPDH by Immunoblotting. (G, H) Densitometry analysis of Western blots shows quantitation of pSTAT3 and pERK levels. (I) 4T1.2 cells were serum starved for 12 hours and stimulated with CXCL12 (100 ng/ml) for different time points, as indicated, and incubated at 37°C. After treatment, cells were washed, lysed, and analyzed for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), and ERK with immunoblotting. (J) Densitometry analysis of Western blots shows quantitation of pSTAT3 and ERK levels, *P < 0.05, **P < 0.01, **P < 0.001 versus none, and ^##P < 0.05, ^##P < 0.01 ^###P < 0.001 versus control.