The pellet was resuspended in 12. 5 ul of ice cold nuclear extraction buffer an

The super natant contained 30 PFU mL of HCMV at 4, 6, 8 dpi, while no plaque forming virus was detected at 2 dpi. HCMV infection オーダー ABT-888 induces apoptosis in iPSC derived NSPCs To examine whether HCMV infection in NSPC iPSCs induces apoptotic responses, we performed the TUNEL assay combined with IFA using an antibody specific to HCMV gB. As shown in Figure 3A, NSPC iPSCs ex pressing gB was positive for TUNEL staining and those without gB expression was consistently negative. We also performed IFA to analyze the activation status of caspases using antibodies specific to the activated forms of caspase 3 and caspase 9. The results show that the activated forms of caspase 3 and caspase 9 were specif ically detected in more than 80% of HCMV infected NSPC iPSCs expressing IE1 IE2 proteins, but not in mock infected NSPC iPSCs.<br><br> To see whether mitochondrial dysfunction is involved in the activation of caspase 9, intracellular distribution of cytochrome c was analyzed in HCMV infected cells by IFA. As shown in Figure 3D and 3E, buy Afatinib strong signals of cytochrome c were detected in the cytosol of cells ex pressing IE1 IE2 proteins, while only faint signals of cytochrome c were detected in cells not expressing IE1 IE2 proteins or in mock infected cells. These results in dicate that HCMV infection of NSPC iPSCs activated apoptotic responses involving release of mitochondrial cytochrome c and serial activation of caspases. Unfolded protein response in HCMV infected NSPC iPSCs The unfolded protein response, induced by the accumulation of improperly folded proteins within the ER lumen, is associated with multiple cellular responses such as neurodegeneration and apoptosis.<br><br> ER stress sensor molecules, such as PKR like eukaryotic ini tiation factor 2a kinase and inositol requiring enzyme 1, are activated on UPR and engage downstream signaling pathways. To examine whether the caspase 9 activation in HCMV infected NSPC iPSCs is associated with UPR, we analyzed phosphor ylation status of IRE1 and its オーダー AG-1478 downstream target c Jun NH2 terminal kinase in immunofluorescence assays. Both IRE1 and JNK were specifically phosphorylated in HCMV infected NSPC iPSCs, but not in mock infected NSPC iPSCs. In concordance with the previous reports that activated IRE1 catalyzes the non conventional splicing of the mRNA encoding X box binding protein 1, the spliced XBP1 mRNA increased gradually after HCMV infection in NSPC iPSCs.<br><br> We also analyzed phosphorylation status of an other sensor molecule PERK, an ER associated serine threonine protein kinase, and its downstream target the alpha subunit of eukaryotic initiation factor 2. Phos phorylated forms of PERK and eIF2 were specifically detected in HCMV infected NSPC iPSCs, but not in mock infected NSPC iPSCs. The transcription factor activating transcription factor 4, that is preferentially translated on activation of PERK, induces the expression of C EBP homologous protein, a transcription factor with proapoptotic functions. In accordance with these pre vious findings, the mRNA level of CHOP increased grad ually after HCMV infection in NSPC iPSCs. These results suggest that UPR is involved in the activation of caspase cascade leading to apoptosis in HCMV infected NSPC iPSCs.