P eif2α

Proteins were extracted with cell lysis buffer containing 1% Triton X-100, sonicated using QSonica-Q800R2, quantified using bicinchoninic acid assay (Sigma) and separated on NuPAGE™ 4-12% Bis-Tris precast gels (Thermo Fisher). Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals. Images were captured using a ChemiDoc™ Touch Imaging System and processed using ImageLab™ Software.

Cells or fresh tissues were lysed in RIPA lysis buffer with mixture of protease inhibitors (Beyotime #ST506) and PhosSTOP (Roche #04906845001). 30 μg total proteins were subjected to 12% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred to PVDF membranes, which were then blocked with 5% milk for 2 hours. The membranes were then probed with primary antibody for IRE1 (Abcam # ab37073), CHOP (Cell Signaling Technology # 2895), GAPDH (Santa Cruz #sc32233), GRP78 (Abcam # ab32618), p-eIF2α (Cell Signaling Technology # 9721), eIF2α (Cell Signaling Technology # 3597), DUOX1 (Abcam #ab78919), p-PERK (Cell Signaling Technology #3179), PERK (Cell Signaling Technology #3192) at 4°C overnight, and then the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Santa Cruz # sc-2030) for 1.5 hours and finally washed and visualized using Chemiluminescent ECL reagent (Beyotime # P0018).

Harvested liver tissues and cells were lysed with mammalian lysis buffer containing phosphatase and protease inhibitors. Equal amounts of cell lysates were measured with the BCA protein assay reagent (Pierce Biotechnology, Rockford, IL). Lysates were boiled in sample buffer containing β-mercaptoethanol for 5 min. Proteins were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE healthcare). After blocking with 5% skim milk in PBS, membranes were incubated with appropriate dilutions of antibodies at 4°C overnight as follows: HO-1 (Enzo, ADI-OSA-150, 1 : 1000 dilution), PGC-1α (Abcam, ab72230, 1 : 1000 dilution), COX III (Abcam, ab110252, 1 : 20000 dilution), COX IV (Cell Signaling, #4844S, 1 : 1000 dilution), p-eIF2α (Cell Signaling, #9721S, 1 : 1000 dilution), and β-actin (Cell Signaling, #4967S, 1 : 2000 dilution). Membranes were then washed with 0.05% PBS-Tween 20 and incubated with a 1/5000 dilution of HRP-conjugated secondary Abs at room temperature for 1 h. Immunoreactivity was detected by using the ECL detection system (GE Healthcare). Films were exposed at multiple time points to ensure that the images were not saturated. The relative band density was analyzed by using ImageJ software (US National Institutes of Health, Bethesda, MD).

U266 and U937 cells (1 × 10⁶ cells/mL) were treated with indicated concentrations of SM (25 or 50 µg/mL) for 24 h. Then, the cells were lysed with RIPA buffer (1 M EDTA, 1 mM Na₃VO₄, 1 mM NaF, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid) containing a protease inhibitors cocktail (Amresco, Solon, OH, USA). The protein supernatant was collected and quantified for protein concentration by using an RC DC protein assay kit II (Bio-Rad, Hercules, CA, USA). The proteins (30 µg) were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes that were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 and incubated with the required antibodies. Primary antibodies, including cleaved PARP (1:1000, 89 kDa), CHOP (1:500, 27 kDa), P-eIF2α (1:500, 38 kDa) (Cell Signaling, Beverly, MA, USA), P-ATF4 (1:500, 39 kDa) (Thermo Fisher, Waltham, MA, USA), P-PERK (1:500, 125 kDa) (Thermo Fisher, Waltham, MA, USA), c-Jun (1:1000, 39 kDa), and β-actin (1:1000, 43 kDa) (Santa Cruz, Dallas, TX, USA), were used at a 1:500~1000 dilution (5% bovine serum albumin) and secondary antibodies at a 1:1000 dilution (5% skim milk) (Santa Cruz, Dallas, TX, USA). After detected protein bands were visualized by Hybond ECL (Amersham Pharmacia, Piscataway, NJ, USA), the blots were scanned.

Western blot analysis was performed as previously described [36 (link)]. Primary antibodies against PCNA and SREBP1C were from Santa Cruz Biotechnology, Santa Cruz, CA; antibodies against FASN were from BD Biosciences, Franklin Lakes, NJ; antibodies against cleaved-caspase 3, cleaved-caspase 8, cleaved-caspase 9, p-eIF2α, t-eIF2α and GCN2 were from Cell Signaling Technology, Beverly, MA.

The rabbit polyclonal antibodies directed against pEGFR, EGFR, pEIF2α, EIF2α and GAPDH and the mouse monoclonal antibody directed against ATF4 were all obtained from Cell Signaling Technology (Danvers, MA, USA). The RCN1 antibody was from Abcam (Cambridge, UK). The mouse monoclonal ATF6 antibody was from Enzo Life Sciences (Redfern, NSW, Australia). The EGFRvIII (LMH-144) antibody was generated in house at the Olivia Newton John Cancer Research Institute. Tunicamycin, thapsigargin and temozolomide were all purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). EGF was purchased from Life Technologies (Life Technologies; Carlsbad, CA, USA) and the anti-EGFR inhibitors: Erlotinib and Gefitinib were obtained from Selleck Chemicals (Houston, TX, USA). The Luciferase Reporter Assay reagents were purchased from Promega (Madison, WI, USA). Human RCN1 and negative control siRNA were from ThermoFisher Scientific (Scoresby, VIC, Australia).

B-MD-C1 (wt) was purchased from ATCC, DOX (Adriamycin, ADR) was purchased from Hisun pharmaceuticals, Zhejiang, China and dissolved in sterile saline solution (2 mg/ml) and stored at -20 °C before use. Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Gibco, USA; Lipofectamine™2000 was purchased from Life Technologies, USA; antibodies against CRT, ERp57, caspase-8 and cleaved-caspase-8 were purchased from Abcam, USA; antibodies against p-PERK, eIF2α and p-eIF2α were purchased from Cell Signaling Technology, USA; β-actin and horseradish peroxidase-labeled antibodies were obtained from Santa Cruz, USA. MTT assay kit was obtained from Amresco, USA.