Fig. 2. POSH acts upstream of the MLK family, MKK4/7 and c-Jun in the JNK pathway and in a neuronal death pathway. (A) POSH overexpression-induced death is significantly suppressed by dominant-negative forms of MLK family members, MKK4, MKK7 and c-Jun. pCMS.EGFP or POSH in the latter vector were co-transfected with either pCDNA3, d/n MLK family members (MLK1, 2 and 3, and DLK), d/n MKK4 or d/n MKK7 in the pCDNA3 vector. Three days after transfection, the levels of survival and the proportions of apoptotic nuclei were determined. The values are the means of three independent experiments ± SEM. The results from co-transfection of pCMS.EGFP/pCDNA3 and pCMS.EGFP with d/nMLKs and d/n MKK4 or d/n MKK7 were not apparently different from each other, and hence only results with pCMS.EGFP/pCDNA3 are shown. (B) Death induced by POSH expression is suppressed by the JNK pathway inhibitor, CEP-1347, and the pan-caspase inhibitor, BAF. pCMS.EGFP and POSH in pCMS.EGFP vector were transfected into neuronal PC12 cells. Half of the cultures were pre-treated and maintained with either 25 µM BAF or 200 nM CEP-1347. Two days after transfection, cell numbers and percentages of apoptotic nuclei were determined. Numbers of cells transfected with pCMS.EGFP, pCMS.EGFP/BAF or pCMS.EGFP/CEP-1347 were each defined as 100%, and values from the other transfections were normalized to them accordingly. Values are the means of three independent experiments ± SEM. (C) POSH and MLKs are synergetic in induction of the JNK pathway. 293 cells were transfected with pCMS.EGFP or POSH, MLK3, DLK, d/nMLK3 or d/nDLK in the same vector, either alone or in combination as indicated. For the cultures transfected with pCMS.EGFP alone, 3 µg of DNA was used while, for all others, the amount of each construct used was 1.5 µg. CEP-1347 (200 nM) was added where indicated 4 h after transfection. Cell lysates were analyzed by western immunoblotting for levels of phospho-JNK. The membrane was stripped and re-probed with EGFP antibody as a transfection control and JNK as loading control. (D) POSH and MLKs are synergetic in the induction of apoptosis in neuronal PC12 cells. Neuronal PC12 cells were transfected with 0.5 µg of pCMS.EGFP or POSH, MLK3 or DLK in the same vector, with either 0.5 µg of pRK5 or pRK5-POSH in the presence or absence of 200 nM CEP-1347 as indicated. Two days later, the numbers of transfected cells were evaluated. Values are the means of three wells ± SEM. Similar results were obtained in two additional independent experiments. (E) Apoptotic death induced by POSH is suppressed by myristylated AKT (MyrAKT). pCMS.EGFP as well as POSH or DLK in the pCMS.EGFP vector were co-transfected with either pCMV6 (control) vector or pCMV6.MyrAKT (MyrAKT). Three days after transfection, the numbers of surviving transfected cells were counted. Cell numbers for pCMS.EGFP/pCMV6 and pCMS.EGFP/pCMV6.MyrAKT were defined as 100% survival and the cell numbers for other transfections were normalized accordingly. Values are the means from three independent experiments ± SEM.