Gsk2656157

The primary antibodies used are listed as follows: anti-cytochrome c (1:1,000, 10993-1-AP, Proteintech, rosemont, IL, USA), anti-caspase-9 (1:1,000, #9508, Cell Signaling, Danvers, MA, USA), anti-PERK (1:1,000, #3192, Cell Signaling, Danvers, MA, USA), anti-phosphorylated-PERK (anti-p-PERK; Thr982;1:800, DF7576, Affinity Biosciences, Cincinnati, OH, USA), anti-IRE1 (1:500, ab37073, Abcam, Cambridge, MA, USA), anti-p-IRE1 (phosphor S724; 1:1,000, ab48187, Abcam, Cambridge, MA, USA), anti-activating transcription factor 6 (anti-ATF6; 1:1,000, ab203119, Abcam, Cambridge, MA, USA), anti-N’ATF6 (1:800, ab37149, Abcam, Cambridge, MA, USA) and anti-β-actin (1:3,000, GTX109639, GeneTex, Irvine, CA, USA). The ER stress activator Tunicamycin (TM) and the p-PERK inhibitor GSK2656157 were obtained from MedChem Express (Monmouth Junction, NJ, USA). The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).

To mimic hypoxic-ischaemic injury, an oxygen-glucose deprivation (OGD) model (O₂ < 0.1%) was constructed by AnaeroPack (Cat# D07, Mitsubishi Gas Chemical Company, Japan) in a 2.5 L closed plastic box. After reaching 70–80% confluency at 3 days, OPCs were incubated in hypoxic conditions with glucose-free DMEM (Cat# 11966025, Gibco) for 3 h and then switched to DMEM-F12 containing 10% FBS and 1% penicillin/streptomycin. The inositol-requiring protein 1α (IRE1α) inhibitor STF083010 (Cat# 307543-71-1, MedChemExpress) (Papandreou et al., 2011 (link); Zhao et al., 2017a (link)) and protein kinase RNA-like ER kinase (PERK) inhibitor GSK2656157 (Cat# 1337532-29-2, MedChemExpress) (Atkins et al., 2013 (link)) with different concentrations were used to treat primary oligodendrocytes at 1 h before and after OGD stimulation, respectively. Then, OPCs were collected for quantitative real-time PCR (RT–PCR), and live/dead cells by Calcein AM/PI double staining and flow cytometry of apoptosis by Annexin V-EGFP/PI double staining (Cat# KGA103, Jiangsu KeyGen BioTech Corp., Ltd. China). Staining methods followed the reference specification. Three independent experiments were performed.

After seeding N2a cells for 24 h, cells were pretreated with 4 µM GSK2656157 (MedChemExpress LLC, Shanghai, China) (a PERK inhibitor) and 5 µM Z-ATAD-FMK (BioVision Inc., Milpitas, CA, USA) (a caspase-12 inhibitor) for 1.5 h and 6 h, respectively. Following pretreatment with inhibitors, Lipofectamine 3000 reagent was used to transfect cells with plasmids as described previously.