These data suggest an important role for JNK1 in innate immune responses

e. U87 cells, which do not express PTEN as they have mutations in both PTEN alleles, acquired PTEN expression. doi:10.1371/journal.pone.0070047.g002 a control to show the ability of exosomes to compensate for PTEN downregulation in DU145Kd. This procedure was used for U87 and PC-3 cells. Prostate Cancer Patients and Normal Subjects This study is Acacetin supported by ethical approval from the McMaster University ethical board. The participants were informed about the purpose of the study, and written consent has 5 Exosomal-PTEN in Prostate Cancer 6 Exosomal-PTEN in Prostate Cancer after surgery and snap frozen in liquid nitrogen. Clinical records such as Gleason score, PSA, tumor size, tumor histopathological grade and metastasis were collected. Samples were collected according to the McMaster University ethical standards, and 11182320 patient consent was obtained before sampling. Eight healthy volunteer men aged 5065 were used in this study; all were without any history of cancer and with normal PSA levels. Statistical Analysis All experiments were reproduced at least three times with similar results. The quantitative data are presented as the average value of the replicates within the representative experiment 6 SEM. Statistical significance was evaluated using a computerized 2-tailed Student’s t-test. The differences were considered significant at P,0.05. Results PTEN is Expressed in Exosomes from PC Cells, but not Normal Cells We determined the status of PTEN in the following PC cells: DU145, DU145 stably transfected with PTEN siRNA for PTEN downregulation or knockdown, and DU145 transfected with control siRNA. Exosomes were collected from the conditioned media of each cell type, and equal protein concentrations from the cells and the exosomes were subjected to SDSPAGE and immunoblotting, then probed with PTEN antibodies. Both DU145Kd cells and exosomes showed a downregulation of PTEN expression compared to the wild type DU145 cells and DU145 cells transfected 7528253 with control siRNA. We also assessed the phosphorylation status of PTEN in the exosomes derived from these cells. Phosphorylation of PTEN keeps it in a closed state protected from degradation; later PTEN will be available to be activated in the cytoplasm, bind the cell membrane upon dephosphorylation, and then initiate cell signaling. We found that the PTEN incorporated in exosomes is phosphorylated. Flotilin-1 was used as a loading control for exosomes. The proliferation rates of DU145 parental cells, DU145 with control siRNA and DU145Kd cells show that the PTEN knockdown cells exhibited a significantly higher proliferation rate than the other two cell types. Human primary cells, such as Human Aortic Endothelial Cells, Human Aortic Smooth Muscle Cells, and Human Prostate Epithelial Cells express PTEN, but we found that PTEN is not incorporated into the exosomes of these cells. This may mean that the incorporation of PTEN in exosomes is an exclusive characteristic of cancer cells, however this finding requires further exploration. Intercellular Transfer of PTEN by Exosomes To investigate the intercellular exchange of PTEN, exosomes derived from native DU145 PC cells were collected using the standard procedure of centrifugation. DU145Kd cells were incubated with the exosome preparation derived from parental DU145 cells. The apparent uptake of microvesicular PTEN by DU145Kd cells was observed using immunocytochemistry, and immunoblotting. been obtained from all individuals who participated in the