A Flow Cytometric Study of ER Stress and Autophagy

AbstractThe mechanistic link between ER stress, autophagy, and resultant cell death was investigated by the use of drugs Thapsigargin (Tg) and Chloroquine (CQ) with prior induction and or blockade of autophagy and apoptosis which modulated the ER stress response and resultant form of cell death. All these biological processes can be measured flow cytometrically allowing the determination of the type of cell death, G1 cell cycle arrest, cell cycle dependent measurement of ER stress transducer PERK, misfolded proteins, reticulophagy, and autophagy marker LC3B. Jurkat cells after Tg or CQ treatment became necrotic and apoptotic, showed G1 cell cycle arrest, autophagy, and ER stress. Prior induction of autophagy before ER stress increased levels of necrotic and apoptotic cell death. Autophagy was further up ‐regulated, while PERK was reduced or abrogated. CQ showed reduced levels of misfolded proteins and reticulophagy, while Tg showed no change in misfolded protein levels but increased reticulophagy and thus displayed more ER stress. Prior blockade of apoptosis before induction of ER stress resulted in cell survival except with high Tg levels which induced necrosis. Autophagy was up‐regulated with modulation of PERK and reticulophagy levels with an abrogation of the misfolded protein response. Blockade of apoptosis with induction of autophagy before ER stress showed death by necrosis with hi gh dose drugs and cell survival with low doses of drugs. CQ induced reduced levels G...

Source: Cytometry Part A - November 19, 2018 Category: Molecular Biology Authors: A Popat, AA Patel, G Warnes Tags: Original Article Source Type: research