2D). More important, HCC cells overexpressing Cryab had a mesenchymal phenotype in HCC tissues (Fig. 2E). Thus, we conclude

that the Cryab overexpression promotes HCC progression by inducing cancer cell EMT. We employed a multivariate approach for integrating genome-wide expression data and biological knowledge23 to search for pathways that are dysregulated as a consequence of Cryab overexpression. We used this method to search through functional pathways defined by KEGG see more and BioCarta in Hep3B-Mock versus Hep3B-Cryab cells. Pathways with significant P values at 0.01 are shown. We identified 28 gene sets in the BioCarta database and 67 gene sets in the KEGG database, with false discovery rates (Q values) < 0.05 by paired t tests between these populations (Table S5). The magnitudes of MEK, FAK, Src, p38, ERK1/2, p65, and Akt phosphorylation

in Hep3B-Mock and HCCLM3-Mock cells were compared to their corresponding control cells. As shown in Fig. 3A, markedly elevated levels of MEK, ERK1/2, and p38 phosphorylation were detected in HCCLM3-Mock and Hep3B-Cryab cells compared with the corresponding control cells, while consistent changes in Src, FAK, and p65 phosphorylation were not observed in the Hep3B-Cryab/Hep3B-Mock Alpelisib price and HCCLM3-Mock/HCCLM3-vshCryab cells. Immunofluorescent staining showed that expression of Cryab in Hep3B-Cryab and HCCLM3-Mock cells correlated with high ERK1/2 phosphorylation (Fig. 3B). To identify the signaling pathways that might contribute to the observed phenotypic changes, we blocked MEK/ERK signaling using U0126 or P38 signal using SD203580. Upon ERK1/2 inhibition, the Hep3B-Cryab and HCCLM3-Mock cells presented an epithelial phenotype based on mRNA and protein expression (Fig. 3C)

and cellular characteristics, such as decreased E-cadherin and increased Fn 1, vimentin, and F-actin when compared with the parental cells (Fig. 3D). These results indicate that hyperactivation of ERK1/2 appears to be crucial for the observed Cryab-mediated phenotypic characteristics of HCC cells. Sorafenib, an oral multikinase 上海皓元医药股份有限公司 inhibitor, offers hope for the clinical treatment of several advanced solid cancers by inhibiting intracellular signals in the ERK cascade and blocking receptor tyrosine kinases.17, 24 Here, we tried to assess whether sorafenib inhibited the activity of the ERK cascade induced by Cryab overexpression. As shown in Fig. S3, Hep3B-Mock cells were evidently inhibited compared with Hep3B-Cryab cells when the sorafenib concentration reached 10 or 20 μM (P < 0.05). We further evaluated the changes in the phosphorylation levels of ERK1/2 by western blot in both Hep3B-Mock/Hep3B-Cryab and HCCLM3-Mock/HCCLM3-vshCryab cells after treatment with sorafenib in a dose-dependent or time-dependent manner at the concentration of 10 μM.