P perk

Total protein was extracted from HL‐60 cells and mice tumours, and protein concentration was quantified by the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The protein samples were separated by 7.5%‐12% SDS‐PAGE gels (Applygen, Beijing, China) and transferred onto polyvinylidene fluoride membranes (120 minutes at 100 V) using standard procedures. The membranes were blocked in TBST (PBS containing 0.1% Tween) containing 5% non‐fat dry milk for 120 minutes at room temperature and then incubated with primary antibodies against CRT (1:8000; Abcam), C/EBPα (1:500; Abcam), PERK (1:1000; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), p‐PERK (1:1000; Santa Cruz Biotechnology Inc), AKT (1:8000; Abcam), p‐AKT (1:1000; Santa Cruz Biotechnology Inc), and GAPDH (1:1000; Santa Cruz Biotechnology Inc) overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibody (1:3000; Anti‐rabbit IgG, HRP‐linked Antibody; Abcam) for 1 hour at room temperature. Membranes were then extensively washed and developed using an enhanced chemiluminescence detection system (CWC, Beijing, China).

Primary antibodies targeting the following proteins were used in this study: p-eIF2α, IRE-1α, CHOP (Cell Signaling Technologies, Danvers, MA, USA), GRP78, ATF6α, PERK, p-PERK, sXBP-1, β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), p-IRE-1α (Abcam, Cambridge, MA, USA) and monoclonal PDI (clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. IC87114 was obtained from Calbiochem (San Diego, CA, USA). Ovalbumin, lipopolysaccharide, N-ethylmaleimide (NEM), 4-hydroxy-2-nonenal (4-HNE) and dihydroethidine (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Propidium iodide (PI), N-acetyl-L-cysteine (NAC), DCFH-DA (6-carboxy-2,7-dichlorodihydrofluorescein diacetate), and PEITC were purchased from Sigma Aldrich (St. Louis, MO, USA) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-­diphenyltetrazolium bromide) from Amresco (Solon, OH, USA). Fluorescein isothiocyanate (FITC)-labeled annexin V (Annexin V-FITC) kit was obtained from BD Biosciences Pharmingen (San Diego, CA, USA). Antibodies against ATF-6 and IRE1α were purchased from Cell Signaling Technology (CA), P-PERK, CHOP/GADD153, and poly-ADP ribose polymerase (PARP) from Santa Cruz Biotechnology (CA), BiP/GRP78 from BD Transduction Laboratories^TM, and GAPDH from AbFrontier (Seoul, South Korea).