Effects of various inhibitors on protrusion formation of MKN-1 cells in 3D collagen gel.

<p>MKN-1 cells were incubated with 10 ng/ml TGF-ß in serum-containing medium in 3D collagen gel culture, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053209#pone-0053209-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053209#pone-0053209-g006" target="_blank">6</a>. To the collagen culture were added various inhibitors, and the protrusion formation was quantified 24 h later (A, C and D) or 3 h later (B). (A) The indicated concentrations of cytochalasin B and colchicine were added into the culture medium at the same time as the TGF-ß addition. (B) Cytochalasin B (5 µM) and colchicine (1 µM) were added into the culture medium after incubation with TGF-ß for 24 h. (C) MKN-1 cells were treated without (Control) or with cantharidin (1 µM) and/or radicicol (1 µM) as described in (A). (D) MKN-1 cells were pretreated with 10 µg/ml of each neutral antibody at 37°C for 30 min, and the pretreated cells were embedded into collagen gel containing 10 µg/ml of the indicated anti-integrin antibody and 10 ng/ml TGF-ß. All these inhibitors were not cytotoxic at least under the above experimental conditions, as analyzed by the trypan blue staining. However, cytotoxic effects became evident when the cells were incubated with 5 µM cytochalasin B or 1 µM colchicine for 24 h.