Figure 1.

CDCP1 protein expression in colon cancer cell lines and modulation of adhesion and motility. A) Cell-surface expression of CDCP1 was measured by flow cytometry using monoclonal mouse anti-CDCP1 clone CUB1. Bound primary antibody was detected with Alexa488-conjugated goat anti-mouse IgG. Standard error bars are shown. n ≥3, except HaCaT where n =2. Cell lines are indicated that showed significantly different CDCP1 cell-surface expression from HaCaT as determined by independent sample 2-tailed t-tests not assuming equal variance. GMF CDCP1, geometric mean fluorescence of bound anti-CDCP1. *p ≤0.05; *** = p ≤0.001. B) Western blot for CDCP1. Total cell protein (40 μg) was separated by SDS-PAGE, transferred to PVDF and CDCP1 detected using 1 μg/ml goat polyclonal antibody anti-CDCP1 antibody (ab1377). The membrane was re-probed for GAPDH to assess protein loading. The image is representative of three blots from independent lysates. C) Binding of stable CDCP1-expressing Colo320 (Colo320-CDCP1) and control Colo320 (Colo320-pcDNA3) cells to control or 10 μg/ml Matrigel coated tissue culture plates. The Colo320 cell-substratum assays were repeated four times. *** = p ≤0.001. D) Adhesion of SW480 cells to control or Matrigel-coated tissue culture plates following reduction of CDCP1 by RNA interference. The experiment was repeated three times. Control, cells transfected with control siRNA; CDCP1, cells transfected with CDCP1 siRNA oligo 2 (see Additional file 1: Figure S2). E) Motility of SW480 cells through an 8 μm pore membrane towards serum following reduction of CDCP1 by RNA interference. This was measured in arbitrary units (AU) at 60 h after siRNA transfection using the xCELLigence system Real-Time Cell Analyzer Dual Plate (RTCA DP, Roche). UT, untransfected cells; mock, cells treated with Lipofectamine 2000 only; Control siRNA, cells transfected with CDCP1 siRNA oligo 2. See Additional file 1: Figure S3 for a more extensive kinetic analysis.