Fig. 2

MLN affects distinct sets of membrane proteins in PCa cells. A Effect of MLN on protein expression in PCa cells measured by western blotting. The numbers indicate the fold increase compared to the control. B Western blots show upregulation of TJ proteins in LNCaP and VCaP cells with the corresponding quantification below. C Effect of MLN on ItgB1 and related signaling in PC3 cells. D Cell morphology and expression of Cldn4 and Ocln proteins in LNCaP and VCaP cells treated with 100 nM MLN. Scale bar = 25 µm. E Expression of ItgB1 in PC3 cells treated with 100 nM MLN. Actin-positive protrusions in MLN-treated cells showed no ItgB1 staining compared to the control (white arrows). Scale bar = 25 µm and 10 µm (zooms). F On the left: binding of Cy3-labeled C-CPE to control and 100 nM MLN-treated LNCaP cells analyzed by flow cytometry. The gray trace corresponds to the control cells not exposed to Cy3-C-CPE. In the middle: effect of 200 µg/ml C-CPE on wound closure by control and 100 nM MLN-treated LNCaP cells. The C-CPE conditions are shown in dark gray. On the right: effect of 200 µg/ml C-CPE on spheroid assembly by control and 100 nM MLN-treated VCaP cells. The C-CPE conditions are shown in dark gray. G Effect of 100 nM MLN on PC3 cell morphology measured by automated microscopy. In all experiments, the cells were treated with MLN for 20 h with DMSO as a vehicle control. Statistical significance: *- p < 0.05, **-p < 0.01 and ***-p < 0.001