Figure 4

Effects of γ-tocotrienol (γT³) and/or the Met tyrosine kinase inhibitor, SU11274 (SU), on the expression of major epithelial and mesenchymal cellular protein markers in + SA mammary tumor cells. Cells were incubated with control or treatment media containing subeffective doses of γ-tocotrienol (2 μM) and SU11274 (3 μM) either alone or in combination containing 10 ng/ml HGF as a mitogen for 3-days. (A) Afterwards, whole cell lysates were prepared and subjected to polyacrylamide gel electrophoresis and Western blot analysis for E-cadherin, β-catenin, cytokeratin-8, cytokeratin-18, and vimentin. α-Tubulin was visualized to ensure equal sample loading in each lane. Scanning densitometric analysis was performed for each blot to visualize the relative levels of proteins. Integrated optical density of each band was normalized with their corresponding α-tubulin and control treatment bands and shown in bar graphs. Vertical bars indicate the fold-change in protein levels in various treatment groups ± SEM as compared with their respective vehicle-treated control group. *P < 0.05 as compared to their respective vehicle-treated control group. (B) Treatment effects on immunocytochemical fluorescence staining of epithelial and mesenchymal markers in + SA mammary tumor cells after a 3-day culture period. +SA cells were seeded on 4-chamber culture slides at a density of 1x10⁵ cells/chamber (3 replicates/group) and allowed to attach overnight. Cells were then washed with PBS and incubated with vehicle control or treatment defined media containing 10 ng/ml HGF for 3 days in culture. At the end of treatments, cells were fixed with 4% formaldehyde/PBS and permeabilized with 0.2% triton X-100. Fixed cells were blocked and incubated with specific primary antibodies followed by incubation with Alexa Fluor 594- or 488-conjugated secondary antibodies. Red or green color indicates positive fluorescence staining for target proteins, while blue color represents nuclear counter staining with DAPI. Magnification is 200X. Figure obtained from reference 25 with permission.