A Flow Cytometric Study of ER Stress and Autophagy

Abstract

The 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, G 1 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 G 1 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 high dose drugs and cell survival with low doses of drugs. CQ induced reduced levels G 1 cell cycle arrest while it was maintained with Tg. Autophagy was also maintained with reduced levels of ER stress. These data demonstrates a profound link between the processes of ER stress, autophagy, and the resultant form of cell death all of which can be modulated depending upon the sequence and concentration of drugs employed.

Downregulation of calponin 2 contributes to the quiescence of lung macrophages

“…As an actin cytoskeleton-associated regulatory protein, calponin 2 may contribute to immune regulation by participating in the ER stress signaling. To test this possibility, ER stress markers PERK (protein kinase RNA-like endoplasmic reticulum ki- nase) and CHOP (CCAAT/enhancer-binding protein homologous protein) were assessed in macrophages isolated from WT and Cnn2 Ϫ/Ϫ mice by flow cytometry using a previously described method (74,101). Our data indicate that Cnn2 Ϫ/Ϫ peritoneal macrophages exhibit significantly reduced expres-sion of PERK and CHOP (Fig.…”

Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death

“…In this study we have added markers for autophagy (LC3B) and ER stress (PERK) making it possible to detect 248 RCD Jurkat cell populations flow cytometrically [ 4 , 20 – 22 ]. This multiplexing was particularly useful for investigating the mode of action of drugs.…”

An immunogenic cell injury module for the single-cell multiplexed activity metabolomics platform to identify promising anti-cancer natural products

“…A common feature of chemically induced immunogenicity is activation of damage response and cell stress signaling pathways including modulation of autophagy ( 8 , 9 , 11 , 60 ), activation of the PERK arm of the UPR ( 10 , 28 , 60 ), DNA stress ( 12 , 61 ), and certain death modalities ( 48 , 62 , 63 ) ( Fig. 6 ).…”