Xbp1s

VLX1570 was synthesized by OnTarget Chemistry AB (Uppsala, Sweden). BSO, Thapsigargin, Z-VAD-FMK were obtained from Sigma Aldrich (Darmstadt, Germany). Antibodies were from the following sources: ubiquitin Lys48 (Merck, Kenilworth, NJ, USA), active caspase-3, BIP, and XBP1S obtained from Cell Signaling Technology (Danvers, MA, USA). PARP and antiheme oxygenase-1 were obtained from BD Biosciences (San Jose, CA, USA). β-actin and HSP70B were obtained from Sigma-Aldrich (Darmstadt, Germany). USP14 was obtained from Bethyl Laboratories (Montgomery, TX, USA).

The cells were collected and lysed in lysis buffer (1 M Tris–HCl, 5 M NaCl, 1% Nonidet P-40 (v/v), 1% sodium deoxycholate, 0.05% SDS, 1 mM phenylmethylsulfonyl fluoride) for 20 min. After brief sonication, the lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and the protein content in the supernatant was measured using a Bio-Rad Protein Assay kit. The protein lysates were denatured at 96°C for 5 min after mixing with SDS-loading buffer, applied on an SDS polyacrylamide gel (Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan) for electrophoresis, and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Western blot analysis was performed using primary antibodies (1:1000) to CHOP (#5554), BiP (#3183), XBP-1s (#12782), cleaved caspase-8 (Asp391) (#9496), BID (#2002), Bcl-2 (#2876), Bax (#2772), caspase-3 (#9662), p38 (#9212), phospho-p38 (#4631) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich). The secondary horseradish peroxidase (HRP)-conjugated antibodies (1:5000) were purchased from Cell Signaling Technology. The band signals were visualized using a luminescent image analyzer (LAS4000, Fujifilm Co., Tokyo, Japan) with the ECL chemiluminescence system (Amersham Biosciences) [17 (link)].

Proteins were extracted from the exosomes, liver tissue, and AML12 cells using a RIPA lysis buffer (Rockland Immunochemicals Inc., PA, United States) that included protease and phosphatase inhibitors (Thermo Fisher Scientific). First, the proteins were quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, United States), then 50–100 μg of protein were separated using 10% Mini-PROTEAN^® TGX™ Precast Protein Gel (Bio-Rad Laboratories, Hercules, CA, United States) and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, United States). The membranes were blocked using EveryBlot blocking buffer (Bio-Rad Laboratories, Hercules, CA, United States) for 5 min at room temperature (RT) and were then incubated with primary antibodies against CD63, IRE1a (Invitrogen), p-IRE1a (LSBio, Seattle, WA, United States), PI3K p85, p-PI3K, Akt, p-Akt, β-actin, acetyl-CoA carboxylase (ACC), p-ACC, fatty acid synthesis (FAS), and XBP1s (Cell Signaling, MA, United States) for 12 h at 4°C. Subsequently, the membranes were incubated with a secondary antibody (Anti-Rabbit IgG-HRP, Bio-Rad Laboratories, Hercules, CA, United States) for 1 h at RT. Protein levels were determined using enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, United States) and ChemiDoc XRS + (Bio-Rad Laboratories, Hercules, CA, United States).