Salubrinal

Tunicamycin (Sigma), an inducer of UPR, was dissolved in DMSO and used at 10 μg/ml. Salubrinal (Santa Cruz), an inhibitor of UPR was dissolved in DMSO and used at 5 μg/ml. 3-methyladenine (3-MA; Sigma), an inhibitor of autophagy was used at 2.5 mM. Drugs were added to the medium after virus adsorption and the same volume of drug vehicle was added as control in non-treated cells. Protease inhibitors E-64d and pepstatin A (10 μg/ml each; Sigma) were added to cell culture medium 4 h before cells were harvested for western blot analysis (Klionsky et al., 2012 (link)). The viability of cells with or without treatment was tested with CellTiter-Glo Luminiscent Cell Viability Assay (Promega).

DTT (Sigma): 1 mM; sodium arsenite (Sigma): 0.5 mM; salubrinal (Santa Cruz): 40 μM; guanabenz (Sigma); bafilomycin A1 (Santa Cruz): 500 nM; chloroquine (Sigma): 10 μM.

CSC-3436 was resuspended in DMSO. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 3-methyladenine [3-MA (autophagy inhibitor)] and primary antibody β-actin were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Salubrinal (endoplasmic reticulum stress inhibitor), ISP-1 (ceramide inhibitor), primary antibodies cleavage ATG-5 and primary GRP78 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies Beclin-1, LC3-I/II, ATG5-ATG12 complex, p-AMPK, AMPK, p-mTOR, mTOR and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary antibodies, HRP-conjugated Goat anti-Mouse IgG and Goat anti-Rabbit IgG, were obtained from Millipore (Billerica, MA, USA).

The following reagents were used in this study: torin1 (Tocris Bioscience), harringtonine (LKT Laboratories), T-2 toxin (Cayman), spermidine trihydrochloride (Sigma-Aldrich), salubrinal and NSC119889 (Santa Cruz Biotechnology). Sodium ortho-arsenite (in the form of dihydroarsenite, NaH₂AsO₃) was kindly provided by Pavel Ivanov (MSU). 10 mM harringtonine, T-2 toxin, salubrinal and NSC119889 stock solutions in DMSO, 100 μM torin1 in DMSO, 1 M spermidine and arsenite water solutions were prepared and stored at −85 °C.

Sorted naïve or memory T cells (1 × 10⁵ cells) were cultured in a 96-well plate in the presence or absence of 2.5 ng ml⁻¹ recombinant mouse IL-7 (Peprotech, Rocky Hill, New Jersey). Cells were stained with 7-AAD (eBioscience) and analysed by a CyAn flow cytometer (Beckman Coulter). The living cell population was determined as 7-AAD negative. In some experiments, naïve T cells or DP thymocytes were irradiated at 0.5 Gy or cultured with etoposide (Sigma) at 1 μg ml⁻¹. For detection of eIF2α, sorted T cells that were freshly isolated or preincubated at 37 °C for 1–2 h to reduce the endogenous level of eIF2α phosphorylation were lysed in RIPA buffer, and total cell lysate was subjected to Western blotting. In some experiments, salubrinal (Santa Cruz), a PP1/GADD34 specific inhibitor, was added at 10 μM after the preincubation. For Th1 or Th17 cell differentiation, sorted naïve CD4 T cells were cultured for 4-5 days with bone marrow-derived dendritic cells and soluble anti-CD3ɛ mAb (145-2C11) in the presence of IL-12 (for Th1) or IL-6 and TGF-β (for Th17). Intracellular cytokine staining was performed using fluorescence-labelled anti-IFN-γ and anti-IL-17A at 200-fold dilutions (eBioscience).