Snail promotes nuclear ERK activation.

<p>(A) MCF-7 parental cells were stably transfected with constitutively active Snail cDNA utilizing lipofectamine <u>2000</u>, and stable clones selected using G418 and isolated. Expression of Snail, p-ERK and ERK in representative Neo and Snail clones was analyzed by Western blot analysis <u>(top panel)</u> and quantified <u>(bottom panel)</u>. (B) Expression and localization of p-ERK in MCF-7 Neo and MCF-7 Snail <u>cells</u> was analyzed using immunofluorescence. (C) MCF-7 Snail <u>cells were</u> transiently transfected with either control or Snail siRNA for 72 h and cell lysates analyzed for expression of Snail, p-ERK and ERK by Western blot analysis <u>(left panel) and results of Western blots were quantified and graphed (right panel)</u>. (D) Nuclear and cytoplasmic extracts were isolated from MCF-7 Neo and MCF-7 Snail cells. The expression of Snail, p-ERK, ERK, p-Elk-1, Elk-1, p-p90RSK, and p90RSK was analyzed by Western blot analysis. β-actin was utilized as a loading control for Western blot analysis; DAPI was used in immunofluorescence to identify the nuclei. <u>Magnification, 63× oil</u>. Topoisomerase I (Topo I) was utilized as a nuclear marker while GAPDH was utilized as a cytoplasmic marker. Relative protein levels compared to actin was analyzed by Image J analysis using National Institute of Health (NIH) software and plotted. <u>Values were expressed as mean ± S.E.M. Statistical analysis was done using ANOVA and Tukey's Multiple Comparison as Post Hoc (** <i>p</i>≤0.01).</u> Results are representative of experiments performed in triplicate.</p>