Fig. 6

Plerixafor and CXCR4 signaling interact with PDGFRB. a Dasatinib dose-response of Ewing sarcoma cell lines. Cells were grown in standard conditions and treated for 72 h before relative cell numbers were measured by WST-1 colorimetric assay. b Dasatinib pre-treatment (100 nM) for 2 h reduces plerixafor-induced proliferation in A673 and DC-ES-6 CXCR4-low cells. Relative cell numbers were measured by WST-1 assay after 24 h. c Plerixafor induces phosphorylation of PDGFRB in CXCR4-low cells, which is abrogated by dasatinib pre-treatment. Cells were cultured in serum-free medium for 16 h, transferred to standard growth medium supplemented with 10% serum, and pre-treated with 100 nM dasatinib for 2 h. Plerixafor (1 μM) was added for another 1 h before preparation of whole cell lysates for Western blotting. Actin served as loading control. d Inhibition of Gαi-protein signal transduction does not abrogate plerixafor-induced proliferation in CXCR4-high cell lines or sensitize them to dasatinib. TC-32 and CADO-ES1 cells were pre-treated with pertussis toxin (PTX) (500 ng/ml) or dasatinib (100 nM) for 2 h before addition of plerixafor (10 μM) for another 72 h. Relative cell numbers were measured by WST-1 assay. All bar graphs in this figure depict mean ± SD of three independent experiments. e Both Plerixafor and PTX induce PDGFRB phosphorylation in CXCR4-high cells, which is not abrogated by dasatinib. Cells were cultured in serum-free medium for 16 h, transferred to standard growth medium, and pre-treated with PTX (500 ng/ml) and/or dasatinib (100 nM) for 2 h. Plerixafor (1 μM) was added for another 1 h before preparation of whole cell lysates for Western blotting. For phospho-AKT, an additional short exposure is shown for TC-32 cells. Actin served as loading control. All blots of this figure are representative of three independent experiments. f Graphical summary of our findings and hypothetical model of CXCR4 and RTK signaling crosstalk. Arrows indicate activation and blunt ends indicate inhibition