Snail promotes nuclear ERK activation.

(A) MCF-7 parental cells were stably transfected with constitutively active Snail cDNA utilizing lipofectamine 2000, 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 (top panel) and quantified (bottom panel). (B) Expression and localization of p-ERK in MCF-7 Neo and MCF-7 Snail cells was analyzed using immunofluorescence. (C) MCF-7 Snail cells were 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 (left panel) and results of Western blots were quantified and graphed (right panel). (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. Magnification, 63× oil. 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. Values were expressed as mean ± S.E.M. Statistical analysis was done using ANOVA and Tukey's Multiple Comparison as Post Hoc (** p≤0.01). Results are representative of experiments performed in triplicate.