1. αvβ6 functional assays in the β6-1089 cell line using blocking antibodies.
αvβ6 functional assays in the β cell line using blocking antibodies. A, adhesion assay; N-1089 and β cells were incubated with either an IgG isotype antibody (IgG) or an anti-αvβ6 antibody (10D5) then plated onto TGF-β LAP (0.5 μg/mL), PDL (1 μg/mL), or BSA (0.1%) for 45 minutes at 37°C. The cells were fixed and stained with crystal violet, which was solubilized and the absorbance read at 550 nm. The background binding to BSA was subtracted from the PDL and LAP values, and adhesion was normalized to IgG controls. β cells adhesion to LAP was blocked in the presence of the αvβ6-blocking antibody. Error bars show SD of triplicate data, the graph is representative of at least 3 independent experiments. Statistical analysis by the Student t test. P < 0.05 considered significant; ***, < B, migration assay; N-1089 and β cells were incubated with either an IgG antibody (IgG) or an anti-αvβ6 antibody (10D5). The cells were then plated in Transwell chambers, the undersides of which were coated with either BSA (0.1%) or LAP (0.5 μg/mL), and incubated in serum-free conditions for 24 hours. The number of migrating cells was quantified by counting the cells on the underside of the Transwell. Significantly more β cells migrated to LAP than the N-1089 control cell line and this was ablated in the presence of the αvβ6-blocking antibody. Error bars show SD of triplicate data, the graph is representative of at least 3 independent experiments. Statistical analysis by the Student t test, P < 0.05 considered significant; *, <0.05. C, TGFβ luciferase assay; N-1089 and β cells were incubated with no antibody, an IgG antibody (IgG) or an anti-αvβ6 antibody (10D5) and then plated on top of MLEC cells containing a TGFβ-responsive PAI-1 luciferase reporter construct for 16 hours. Cells were lysed and the luminescence read after addition of the substrate. Significantly more luminescence was detected when β cells were plated with the MLEC cells compared with the N-1089 cells; this was suppressed significantly in the presence of the αvβ6-blocking antibody, MLEC cells alone indicate background luminescence. Error bars show SD of triplicate data, the graph is representative of at least 3 independent experiments. Statistical analysis by the Student t test, P < 0.05 considered significant; *, <0.05.
Michael D. Allen et al. Clin Cancer Res 2014;20:
©2014 by American Association for Cancer Research