FAK immunostaining at focal contacts was also visibly reduced by ST. The early actin changes were not affected by the protein synthesis inhibi tor cycloheximide, consistent with post translational mechanisms. In contrast, vimentin induction was significantly reduced by cycloheximide, suggesting de novo production consistent with an induced EMT pro gram. QRT PCR data confirmed selleckchem Erlotinib the immunohisto chemical results shown in Figure 1 Vimentin and MMP 2 mRNA were upregulated, and E cadherin mRNA down regulated, both to a greater degree by the combined treat ment. EGF alone induced vimentin, whereas ST induced MMP 2 and repressed E cadherin more strongly than EGF alone. These changes were largely apparent by 24 hours, and remained or increased by 72 hours.

The effect of combined EGF and ST on reducing Inhibitors,Modulators,Libraries E cadherin Inhibitors,Modulators,Libraries expression was not seen at 24 hours, but was prominent at 72 hours. Snail1 and Snail2, amongst other E cadherin repressors such as Zeb1 EF1 and Zeb2 SIP1, are transcription fac tors which orchestrate EMT because their induction leads to a cascade of downstream gene expression changes which embody and reinforce the EMT phenotype. As shown in Figure 4B, EGF almost exclusively induced Snail2 at the early stage examined, whereas ST induced Snail1 at this time point. When EGF and ST were combined, at 3 h, the relative expression pattern for Snail family genes was dominated by ST. Some Snail2 was still expressed along with Snail1 at 3 h and 24 h, however by 72 h the expression of Snail1 predominated. Snail2 per sisted as the main Snail gene expressed in EGF treated cul tures, and Snail1 in the ST treated cultures.

The Snail1 downstream effector gene, Zeb1 EF1 followed Snail1 induction, being maximally induced at 72 h in all treatments, but the expression level was greatest in those receiving combined treatment. Increased Zeb1 EF1 was also seen following Snail2 induction in EGF treated cells. Although Inhibitors,Modulators,Libraries Zeb2 SIP1 was somewhat induced by EGF at 24 h and at 72 h, and also by ST at 72 h, it was repressed by EGF ST at all treatment times. Staurosporine and EGF individually induce diverse gene expression Inhibitors,Modulators,Libraries profiles The gene expression programmes induced by ST and by EGF alone were dramatically different over the 72 h period in PMC42 LA cells when examined by MT PCR. Genes induced early by EGF alone were AKT1, CYR61, CLDN4, CD44, CLDN1, CTGF, KRT14, KRT7 and S100A4.

Expression of the last 6 genes in this list persisted at later timepoints whereas genes primarily induced late were CAV1, CD24, CDH5, EGFR and LGALS1. Genes which were repressed early by EGF Inhibitors,Modulators,Libraries included CD24, DCN, EPHB4, KRT18, NLF1, NLF2, SPARC and WT1, with DCN, EPHB4, KRT18, SPARC and WT1 repression persisting at later timepoints. Genes mainly repressed only in the later time period by EGF alone were KRT8, PAX2 and PAX6. Genes induced early by ST differed to those induced by EGF at this selleck bio time point.