Functionally active integrin α_Vβ₃ in cancer cells is required for ATX binding. (A) Flow cytometry detection of cell surface expression of integrin α_Vβ₃ in parental CHO-dhfr⁺ (dhfr⁺, left), CHO-β₃WT (β3WT, middle), and CHO-β₃-Δ744 (β3-Δ744, right) cells. Cells were immunostained with the anti-human integrin α_Vβ₃ monoclonal antibody, LM609 (dark histograms) or isotype control antibody, MOPC21 (open histograms). (B) CHO cell adhesion on ATX in presence of Mn²⁺ (2 mM). Optical microscopy observation of cell adhesion on ATX or OPN (scale bar: 200 μm). (C) Dose-response curves of cell adhesion were generated in the presence of increasing amounts of coated ATX. Data represent the mean of adherent cell/mm² (± SD) of 3 independent experiments performed in triplicate (***P < .001; vs CHO-dhfr⁺ using 2-way ANOVA with a Bonferroni posttest). (D) Inhibition of CHO-β₃WT cell adhesion on ATX and FN with LM609 antibody. BSA was used as a negative control for cell adhesion. Cells were preincubated for 1 hour in the presence of LM609 or MOPC21 antibodies (10 μg/mL). Data represent the mean of adherent cell/mm² (± SD) of 3 experiments performed in triplicate (***P < .001; vs CHOβ₃WT cells treated with MOPC21 antibody using 1-way ANOVA with a Bonferroni posttest). (E) Coimmunoprecipitation of ATX and integrin β₃. Cell lysate from stable transfectants of CHO-dhfr⁺/ATX and CHO-β₃WT/ATX expressing mouse ATX were incubated with LM609 antibody. ATX was immunodetected with the anti-LysoPLD antibody. (F) Soluble ATX inhibits cell adhesion of coated ATX. CHO-β₃WT cells were preincubated for 1 hour in the presence of soluble ATX (x-axis) prior to the adhesion assay. Data represent the mean percentage of adherent cells (± SEM) from 2 independent experiments performed in triplicate (***P < .001; vs CHOβ₃WT cells treated with BSA using 1-way ANOVA with a Bonferroni posttest). (G) Inhibition of CHO-β₃WT cell adhesion with soluble functionally active ATX or soluble lysoPLD-deficient ATX-T209A. Cells were preincubated for 1 hour in the presence of soluble ATX, soluble ATX-T209A, or BSA as control (15 μg/mL). Data represent mean percentage of adherent cells (± SD) of 2 experiments performed in triplicate (***P < .001; vs CHOβ₃WT cells treated with BSA using 1-way ANOVA with a Bonferroni posttest). (H) Cell proliferation of CHO-β₃WT (β3WT) and CHO-β₃-Δ744 (β3-Δ744) cells in response to LPC (1 μM) and recombinant ATX (1 nM), in presence or absence of PF8330 (5 nM) or in response to LPA (1 μM) in serum-free Ham’s F-12K medium. Cell proliferation was assessed by densitometry analysis after cell staining with crystal violet. Data are expressed as mean density (± SD) of 6 replicates and are representative of 3 experiments. (*P < .05; vs CHO-β₃WT using 2-way ANOVA with a Bonferroni posttest).