Fig 1: Plasminogen-induced invasion is externalized form of cytokeratin 8 (eK8)-dependent. (A) Effect of anti-K8 M20 antibody, and the D-A10 and D-D6 monoclonal antibodies (MAbs) on Isreco-1 cell invasion. Real-time analysis by X-CELLigence (n = 3 ± standard error of the mean (SEM)). Histogram representing the integration of the mean invasion curve slopes (triplicate of three wells per condition ± SEM) over 70 h compared to non-treated cells (% of inhibition of invasion), calculated using the Real Time Cell Analyzer-Dual Purpose, RTCA-DP software® (ACEA Biosciences Inc., San Diego, CA, USA). (B) Dose-dependent effect of plasminogen on Isreco-1 cell invasion. Cells were placed for 24 h either in 10% fetal bovine serum (FBS) or in serum-free (SF) medium and resuspended in SF medium containing either no or increasing concentrations of plasminogen (Plg; 5, 50, or 500 nM). Real-time analysis of invasive properties was done using the X-CELLigence technology. Histogram representing the integration of the mean invasion curve slopes (triplicate of three wells per condition ± SEM) over 30 h. AU: Arbitrary Unit. (C) Effect of plasminogen in the presence or absence of D-A10 Fab MAb on Isreco-1 cell invasion. Real-time analysis was done using the X-CELLigence system (n = 3 ± SEM). Histogram representing the integration of the mean invasion curve slopes (triplicate of three wells per condition ± SEM) over 30 h compared to control (SF condition). AU: Arbitrary Unit. Student’s t-test: * p < 0.05. (D) Immunofluorescence (IF) analysis by confocal microscopy of K8 (N-terminal antibody, green signal) and plasminogen (anti-Plg antibody, red signal) localization on permeabilized (left panels) and non-permeabilized (right panels) Isreco-1 cells cultured in the presence or absence of FBS. Scale bar = 10 µm. Hoechst dye was used to counterstain the nucleus (blue signal). The inserts represent negative controls without primary antibodies. Superposition or not of green and red signals is presented on enlarged views of the merge images (right panel). (E) IF analysis by confocal microscopy of K8 (N-terminal antibody, green signal) and urokinase-type plasminogen activator (uPA; anti-uPA antibody, red signal) localization on permeabilized (left panels) and non-permeabilized (right panels) Isreco-1 cells cultured in the presence or absence of FBS. Scale bar = 5 µm. Hoechst dye was used to counterstain the nucleus (blue signal).