Effect of E-cadherin knock-down on the expression of SNAI1 and other transcriptional factors. (A) Sh-PC3 and ShEC-PC3 cells were collected at similar confluency and nuclear/cytoplasmic fractions were prepared and analyzed for SNAI1, β-catenin, and p65 expression by Western blotting. Tubulin and histone H1 were used as loading control for cytoplasmic and nuclear fractions respectively. (B) Sh-PC3 and ShEC-PC3 prostaspheres were collected following centrifugation and cell lysates were prepared and analyzed for SNAI1 expression by Western blotting. (C) Sh-PC3 and ShEC-PC3 xenograft tissues were analyzed for the expression of SNAI1 by IHC. Immunoreactivity score was analyzed in 5 random areas for each tumor tissue and was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). Percentage of SNAI1 positive cells was calculated by counting the number of positive stained cells (brown stained) and the total number of cells at five arbitrarily selected fields from each tumor at 400x magnification. The data shown in the bar diagrams is the mean±SEM of 7–10 samples. *, p ≤ 0.001.