We next sought to determine a mechanism whereby alcohol might be turning on the EMT program. The most studied transcription factor regulating EMT is Snail. One recent study showed that PAK1 phosphorylates (activates) Snail at Ser246 resulting in Snail localization to the nucleus and is required for Snail-mediated promotion of EMT (Yang et al., 2005). To determine whether alcohol promotes EMT through Snail, we performed a time course experiment in which cells were stimulated with alcohol (30min, 1h, 2h, 4h, 6h) and nuclear extracts were tested by western blotting with an Ab specific for Snail Ser246. Cytoplasmic extracts were negative (not shown). As seen in Figure 3A–D, alcohol stimulated significant (p ≤ .05, ANOVA with post Hoc Dunnett test) time-dependent nuclear localization of Snail phosphorylated at Ser246 in Caco-2 cells, MDA-MB-231, MCF-7, and IEC-6 cells.
