A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems

Abstract

The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post‐translational, translational, post‐transcriptional, and transcriptional mechanisms initiated by three ER‐resident sensors, inositol‐requiring protein 1α, activating transcription factor 6α, and PRKR‐like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress in vitro and in vivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.

HIPK2 sustains inflammatory cytokine production by promoting endoplasmic reticulum stress in macrophages

“…Previous studies have demonstrated that endoplasmic reticulum (ER) stress serves an essential role in inflammation and cytokine production by macrophages (6,7). The activation of macrophages by lipopolysaccharides (LPS) leads to the unfolded protein response, which results in the activation of three ER stress markers on the ER membrane: Inositol-requiring enzyme 1α (IRE1α), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) (6).…”

Peptidomimetic‐based identification of FDA‐approved compounds inhibiting IRE1 activity

“…The resulting membranes were incubated with primary antibodies for 16 h at 4 °C, washed with PBS and incubated for 1 h with goat anti‐rabbit secondary antibodies at room temperature (Invitrogen) prior revelation using chemiluminescence. The antibodies used were as previously described [40].…”

Local intracerebral Inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo

“…Clarified lysates were complemented with 20 µl of 5 times reducing Laemmli sample buffer prior heat-denaturation and used in Western blot experiments with the indicated antibodies (at 1/1000 dilutions). For RNA preparation, cells were lyzed using 1ml of Trizol reagent per plate and RNA was extracted according to the manufacturer’s instructions (Thermo Fisher Scientific) and PCR analyses performed using previously described protocols [11].…”