Figure 1.

Peptide 5a prevents the adhesion of TRAMP-C2 cells (αvβ8-positive) to plates coated with an anti-αvβ8 antibody or with a peptide 5a-albumin conjugate and inhibits cell-mediated TGFβ activation. (A) Effect of 5a and 2a on the adhesion of TRAMPC-2 cells to PVC-microplates coated with anti-αvβ8 mAb ADWA-11 or mAb 5A8 (a negative control). Cells were mixed with or without the peptides (100 µM) and analyzed by bright-field microscopy after 2h. Representative photographs (10x magnification) are shown. (B) Adhesion of TRAMP-C2 cells to PVC-microplates coated with a 5a-human serum albumin conjugate (5a-HSA), or with a control conjugate made without peptide (*HSA), after 2 h of incubation. Non-adherent cells were removed; adherent cells were stained with crystal violet and quantified by spectrophotometric analysis (A₅₇₀nm) (mean±SE, n=3). Representative photomicrographs of wells coated with 5a-HSA or *HSA (16.6 µg/ml) are shown. (C) Effect of 5a and 2a on the adhesion of TRAMPC-2 cells to PVC-microplates coated with 5a-HSA (10 µg/ml). Adherent cells were stained with crystal violet and analyzed spectrophotometrically (mean±SE, n=2-3). (D) Effect of 5a and 2a on TGFβ activation by TRAMP-C2 cells. Cells were seeded on cell culture microplates, left to adhere for 3 h and treated with 5a or 2a for 16 h. The amount of active TGFβ in the supernatant was quantified using a bioassay based on HEK-Blue™ TGFβ cells. Cumulative results of two independent experiments are shown (mean±SE, n=4-6 wells). *, P<0.05; **, P<0.01; ***P <0.001 by two-tail t-test.