Figure 6. Effect of siRNA-mediated downregulation of beta-catenin on fibronectin-directed migration, matrigel-invasion, transcriptionally active beta-catenin and fibronectin-induced activation of RAC1 in TNBC cells.

A. BT20, MDA-MB231 and HCC1937 cells were transiently transfected with beta-catenin siRNA for 96 hours. Total levels of beta-catenin were determined by Western blots and compared between siRNA (24, 48, 72 and 96 hours) and control (24, and 96 hours) cells at similar time points. Actin was used as loading controls. Bar diagram shows changes in the relative density (Arbitrary Units). B. MDA-MB231 and BT20 cells were transiently transfected with beta-catenin siRNA for 24 hours and then allowed to migrate on fibronectin in a transwell assay. Migrated cells were stained with phalloidin 555 and DAPI using the cut-outs of transwell membrane for the picture (upper panel). Bar diagram (*P< 0.05) shows a number of cells migrated through transwell membrane (X10). C. MDA-MB231 and BT20 cells were transiently transfected with beta-catenin siRNA for 24 hours and then allowed to invade across matrigel on fibronectin. Bar diagram (*P< 0.05) shows a number of cells invaded across matrigel (X10). D. Active beta-catenin was determined from lysates of MDA-MB231 and BT20 cells which were transiently transfected with beta-catenin siRNA for 24 hours. Beta-catenin levels were used as the reference for the transient transfection of siRNA. Actin was used as loading control. Bar diagram shows changes in the relative density (Arbitrary Units). E. Active RAC1 (GTP-RAC1) and active beta-catenin were determined from lysates of MDA-MB468, which were transiently transfected with beta-catenin siRNA for 24 hours. Active RAC1 (GTP-RAC1) was determined from lysates of MDA-MB468, which were transiently transfected with beta-catenin siRNA for 24 hours. Actin and total RAC1 were used as loading controls. Bar diagram shows changes in the relative density (Arbitrary Units).