Also, these cells adopted a extra clustered arrangement in the cultures and showed a marked improve in formation of adherens junctions with localization of catenin at cell cell interfaces. In contrast towards the A375 ODAM cells, C8161 ODAM cells adopted a larger, far more rounded morphology relative to your spindle form of cells in manage cultures. These cells did not ex hibit circumferential actin cables or catenin arrangement find more information in adherens junctions. Analysis of signal transduction Human melanomas regularly exhibit dysregulation of crucial signal transduction pathways and their po nents, including these in the Ras Raf MEK MAPK and PI3K AKT mTOR pathways, just about every of which constitute central regulators of cell growth, survival, together with other crit ical parameters of oncogenesis Western blot ana lysis of melanoma cell lysates with phospho unique antibodies exposed a marked reduce in AKT activation in ODAM expressing cells evident as decreased phos phorylation on each the Ser 473 and Thr 308 residues associated with AKT activation when overall ranges of AKT protein have been unaffected.
Accordingly, phosphorylation of c Raf a downstream target of AKT was also decreased. Activation of AKT calls for the NXY059 generation of phosphatidylinositol three,4,5 triphosphate by phos phatidylinositol 3 kinase together with mem brane docking of AKT and dual internet site phosphorylation of AKT by phosphoinositide dependent kinase 1 and mTOR Conversely, activation of AKT is antagonized from the PTEN tumor suppressor gene prod uct by way of its PIP3 phosphatase action Prob ing of western blots with phospho precise antibodies for for the observed suppression of AKT activation. There fore we treated cultures with manage and PTEN distinct siRNAs and assayed PTEN amounts and phospho AKT by western blots of lysates prepared 72 hours later on.