Kira6

For the stress+GSK2606414 and GSK2606414 groups, rats were fed GSK2606414 (Millipore, 516535, Burlington, MA, United States) by oral gavage (in vehicle: 0.5% HPMC, 0.1% Tween-80 in water, pH 4.0) at a dose of 10 mg/kg once a day for 7 days. For the stress+ KIRA6 and KIRA6 groups, rats were i.p., injected with KIRA6 (Millipore, 532281, Burlington, MA, United States; in vehicle: 3% ethanol, 7% Tween-80, 90% saline) at a dose of 10 mg/kg once a day for 7 days. Then, the rats requiring restraint treatment were placed in the restrainer with no access to food and water for 8 h (from 8:00 to 16:00) each day. The stress protocol was adapted from a previously described method (Imbe et al., 2012 (link)); the rats could stretch their legs, but could not move within the restrainers. Then, the restricted rats were placed in ice water to swim for 5 min each day. The stress-inducing exercises lasted for 7 days. The control, GSK2606414, and KIRA6 groups rats were left in the cages for the same time without food and water. During the rest period, all rats were provided food and water ad libitum.

G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.

We developed a 384 well formatted TR-FRET assay using LanthaScreen reagents (ThermoFisher) to evaluate the binding of the IRE1α inhibitor KIRA6 (EMD Millipore or MedChemExpress, Monmouth Junction, NJ, USA) to the purified full-length cytoplasmic domain of IRE1α. We utilized a displacement assay format with a Europium-labeled antibody that recognizes the N-terminal His tag on the IRE1α cytoplasmic protein as an energy donor, and an Alexa Fluor™-labelled kinase ligand, 236, as the energy acceptor (ThermoFisher). CTx-0294885, a non-selective, broad spectrum kinase inhibitor, was used as a positive control for the assay (MedChemExpress). After incubating the assay mix consisting of 20 nM his-tagged IRE1α, 2 nM Europium anti-his antibody, and 40 nM 236 tracer, the TR-FRET signal ratio (665 nm/615 nm) was collected using a Perkin Elmer EnVision^® plate reader. Eight point compound dose curves diluted in half logs starting at 10 µM were utilized to generate the data.