Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma(SH-SY5Y)cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.