Whereas Akt phosphorylation has been reported to be downregulated by enzastaurin, likely through an indirect mechanism as Akt is not a direct target of the drug. CUDC-907 However, enzastaurin has also been reported to have little effect on Akt phosphorylation in glioma cells. In the UM cells studied here, Akt phosphorylation was only affected in Mel285 cells by enzastaurin. Interestingly, Nutlin-3 although both Akt and Erk1/2 phosphorylation were decreased by enzastaurin, Mel285 cells, like other GNAQ wild type cells, were less sensitive to enzastaurin in comparison to GNAQ mutated cells where only Erk1/2 phosphorylation was affected. In agreement with sensitivity to enzastaurin, inhibition of Erk1/2 phosphorylation was accompanied by increased p27Kip1 accumulation and decreased expression of cyclin D1, Bcl-2 and survivin in GNAQ mutant cells whereas only survivin was downregulated in Mel285 cells. Furthermore, inhibition of Erk1/2 phosphorylation by MEK1/2 inhibitors increased sensitivity of GNAQ wild type cells to enzastaurin and was associated with similar alterations in the expression of p27Kip1, cyclin D1, Bcl-2 and/or survivin 
to GNAQ mutant cells treated with enzastaurin. Our findings suggest that the suppression of Erk1/2 phosphorylation may be the major contributor to the increased sensitivity of GNAQ mutant UM cells to the antiproliferative action of enzastaurin through altering the expression of p27, ccyclin D1, Bcl-2 and survivn. These observations further support the oncogenic role for GNAQ mutations via activation of MAPK. The signaling pathways downstream of GNAQ are multifold and include activation of the PKC family members. Our results indicate that UM cell lines have varying expression and phosphorylation patterns of PKC isoforms, independent of GNAQ mutational status. The effects of enzastaurin on the expression and phosphorylation of PKC isoforms in UM cells are complex. Additional studies are needed to determine whether GNAQ mutational status influences the effects of enzastaurin on various PKC isoforms and the potential therapeutic ramifications of these effects. Nonetheless, some PKC isoforms were downregulated by enzastaurin in UM cell carrying GNAQ mutations. In particular, the expression and phosphorylation of PKCh, PKCe, and PKCb were reduced by enzastaurin in GNAQ mutated cells. Our functional studies revealed that these PKC isoforms are indeed more critical for growth of UM cells harboring GNAQ mutations than those with wild type GNAQ. Together, our findings suggest that enzastaurin may exert increased antiproliferative action through inhibiting these PKC isoforms in GNAQ mutated UM cells. Inhibition of these isoforms may play a role in enzastaurininduced inhibition of Erk1/2 phosphorylation, since activation of PKCe and PKCbII have been shown to trigger several major signaling pathways including MAPK. In addition, the inhibition of PKCbII by enzastaurin or small interfering RNA decreased Erk1/2 phosphorylation in metastatic hepatocellular carcinoma cells. It is noteworthy that although enzastaurin had little effect in general on the expression and/or phosphorylation of PKC isoforms in GNAQ wild type C918 cells, it did decrease the expression of PKCe and PKCb phosphorylation in another GNAQ wild type cell line Ocm1. However, enzastaurin did not significantly alter Erk1/2 phosphorylation in both cell lines, suggesting other PKC isoforms and/or PKC independent mechanisms for Erk1/2 activation in Ocm1 cells. Complicating this interpretation, Ocm1 cells have been shown to carry the common V600E BRAF mutation that constitutively activates the MAPK pathway. Furthermore, PKCa and PKCd have been reported to activate Erk1/2 in mouse melanoma. Both PKCa and PKCd are expressed in Ocm1 cells.