Snail is correlated with increased p-ERK in breast cancer cells.

<p>(A) Normal immortalized breast epithelial cells (HMEPiC), the adenocarcinoma cell lines MCF-7 and T47-D, and triple negative cell line MDA-MB-231 were utilized to analyze Snail, p-ERK and total ERK protein levels by Western blot analysis. (B) MCF-7, T47-D, and MDA-MB-231 <u>cell lines</u> were utilized to perform a migration assay on collagen. Cells that had migrated through the boyden chamber insert <u>were</u> fixed, stained, counted and graphed. <u>Bars, SD, paired Student's t-test.</u> (C) T47-D and MDA-MB-231 cells were transiently transfected with control siRNA or Snail siRNA and expression levels of Snail, p-ERK, and ERK were determined with Western blot analysis <u>(top panel)</u>. <u>Relative protein levels compared to actin were analyzed by Image J analysis using National Institute of Health (NIH) software and plotted (bottom panel). Bars, SD. Statistical analysis was done using ANOVA and Tukey's Multiple Comparison as Post Hoc (* <i>p</i>≤0.05, ** <i>p</i>≤0.01)</u>. (D) Immunofluorescent analysis <u>was also performed on T47-D and MDA-MB-231 cells transiently transfected with control siRNA or Snail siRNA</u>. β-actin was utilized as a loading control for Western blot analysis; DAPI was used to identify the nuclei in immunofluorescent analyses. <u>Magnification 20X; Inset 40X</u>. Results represent experiments performed in triplicate at least twice independently.