Figure 3

Wnt/β-catenin pathway plays a critical role in the regulation of metastasis-associated phenotypes in TNBC cell line based models. Functional blockade of Wnt/β-catenin pathway following the administration of Wnt-antagonist, WntC59 (10nM) (A), and transient transfection of β-catenin SiRNA (B) caused a decrease in fibronectin-mediated migration (left panel), and invasion (right panel) in MDA-MB231 TNBC cells. Migration assays (scratch assay), and invasion assays were performed on WntC59 treated or β-catenin SiRNA transfected cells. Crystal violet stain was used for the semi-quantification (Olympus DP72; X10) (A). Functional assays (transwell migration assay, and invasion assay) were carried out at 48 hours following transfection. Lysates from cells transiently transfected with scrambled SiRNA, and β-catenin SiRNA at 24, 48, and 72 hours were quantified for β-catenin expression by Western Blot (B Inset). Attenuator of Wnt-beta catenin signaling (sulindac sulfide) substantially abrogated the F-actin cytoskeletal organization in HCC38 TNBC cell line (Cii) as compared to the control (Ci) as well as in MDA-MB231 TNBC cell line (Ciii). Active beta-catenin levels were semi-quantified in arbitrary units (Image J) following Western blot analyses from the clarified MDA-MB231 cell lysates (beta-catenin SiRNA transfected cells and sulindac sulfide treated cells. Upper bar diagrams showed the relative desitometric expressions of beta-catenin, and active beta-catenin. Beta-actin was used as the loading controls (D left panel). Relative luciferase activity (TOP Flash over FOP Flash) measured in MDA-MB231 cells following beta-catenin SiRNA transfection and sulindac sulfide treatment was plotted (three different experiments). Error bars represent standard error of the means (SDs), and statistical significance was determined by paired t-test. *P < 0.05 (D right panel).