Stf 083010

All chemicals and buffers were of analytical grade and purchased from ThermoFisher Scientific (Massachusetts, USA). Palmitic acid (A3803) and fatty acid free bovine serum albumin (BSA, P5585) were obtained from MilliporeSigma, St. Louis, MS. 15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and GSK2606414 (GSK260) were purchased from Cayman, Ann Arbor, MI. SP600125 (JNKi), STF-083010 (IRE1αi, endonuclease inhibitor), Salubrinal (eIF2αi, dephosphorylation inhibitor), U0126 (ERKi, MEK1/2 or ERK1/2 Inhibitor) were obtained from Selleck Chemicals, Houston, TX.

Adipocytes were incubated with 4-phenylbutyrate (4-PBA, 50 nM, Sigma-Aldrich, MO, USA, SM0309) for 12 h. TM (1 µM, MACKLIN, Shanghai, China) was used to construct an ERS model (Sigma-Aldrich, MO, USA); STF083010 (50 nM, Selleck, Houston, TX, USA) and PF573228 (1 μM, Selleck, Houston, TX, USA) were prepared to treat the cells after transfection. IP3R channel was suppressed with 2-Aminoethoxydiphenylborane (2-APB) (50 nM, Abcam, MO, USA).

Chemical hypoxia was induced with Cobalt Chloride (CoCl₂, Merck, Darmstadt, Germany).
ER stress was induced with HA15 (Selleckchem, Zurich, Switzerland), Thapsigargin (THA, Enzo LifeSciences, Lausen, Switzerland), or Tunicamycin (TUN, Enzo LifeSciences, Lausen, Switzerland).
ER stress was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF; Sigma, Darmstadt, Germany), Melatonin (Mel; Sigma, Darmstadt, Germany), STF-083010 (STF; Selleckchem, Zurich, Switzerland), 4μ8C (Selleckchem, Zurich, Switzerland), GSK2656157 (GSK; Selleckchem, Zurich, Switzerland) or Salubrinal (SAL; Tocris Biosciences, Bristol, UK).

The drugs used in this study were prepared as follows. Morphine hydrochloride (Shenyang First Pharmaceutical Factory, China) was diluted in saline (Northeast Pharmaceutical Group, China). Specific IRE1α endonuclease inhibitor STF-083010 (Selleckchem, Houston, TX, United States) and selective PERK inhibitor GSK2606414 (Selleckchem, Houston, TX, United States) were dissolved in 100% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, United States), respectively. STF-083010 (10 or 50 μg), GSK2606414 (10 or 100 μg) or vehicle solution was intrathecal injected 30 min before morphine administration in a volume of 10 μL, respectively, followed by 10 μL of saline to flush the catheter. The doses of STF-083010 and GSK2606414 used in this study were determined according to our preliminary results.

CB-5083 (S8101, Selleckchem, Houston, TX, USA), NMS-873 (S7285, Selleckchem), DBeQ (SML0031, Sigma-Aldrich, Saint Louis, MO, USA), STF-083010 (S7771, Selleckchem,), STF-083010 (SML0409, Sigma-Aldrich), ISRIB (SML0843, Sigma-Aldrich) and mifepristone (M8046, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO) at 50 mM stock solution. Tunicamycin (T7765, Sigma-Aldrich) was dissolved in DMSO at 10 mM stock solution and ISRIB (SML0843, Sigma-Aldrich) was dissolved in DMSO at 5 mM stock solution. All stock solutions were aliquoted in small volumes and stored at −80 °C. Before use, appropriate concentrations of these compounds were prepared by dissolving the stock solution (or its subsequent dilution) into the appropriate growth media.

The IRE1/XBP1s activator IXA4 was custom synthesized by Otava chemicals. The IRE1 RNase inhibitor 4μ8c was purchased from EMD Millipore (catalog no. 412512) and STF-083010 was purchased from Selleck Chemicals (catalog no. S7771). Antibodies used in this study include: phospho-c-jun (Cell Signaling, catalog no. 3270S), phospho-JNK (Cell Signaling, catalog no. 4668S), JNK (Cell Signaling, catalog no. 9252S), phospho-AKT(Ser473) (Cell Signaling, catalog no. 4060S), AKT (Cell Signaling, catalog no. 2920S), FOXO1 (Cell Signaling, catalog no. 2880S), LaminB1 (Cell Signaling, catalog no. 13435S), tubulin (Sigma, catalog no. T6074-200UL), XBP1s (E9V3E) (Cell Signaling, catalog no. 40435S), BiP (Cell Signaling, catalog no. 3177S), SEC24D (gift from William Balch’s lab at Scripps), PERK (Cell Signaling, catalog no 3192S), eIF2α (Cell Signaling, catalog no 9722), phospho-eIF2α (Cell Signaling, catalog no 9721), and PCK1 (Abcam, catalog no. ab70358).