Cycloheximide (chx)

BMDMs and RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% of fetal bovine serum (FBS, Sigma-Aldrich). BMDMs were isolated from C57BL/6 mice as described elsewhere.^{44 (link)} BMDMs and RAW 264.7 cells were pretreated with GPL, N-acetyl-L-cysteine (NAC, Sigma-Aldrich), or cyclosporin A (Sigma-Aldrich) for 2 h, washed, and stimulated with apoptotic factors or infected with MAB at indicated MOIs. The cells were incubated with apoptotic inducers staurosporine (100 nM, Enzo Life Sciences, Farmingdale, NY, USA), H₂O₂ (0.5 mM, Sigma-Aldrich), TNF-α (20 ng/ml, eBioscience, San Diego, CA, USA) plus 2 μg/ml cycloheximide (Enzo Life Sciences), or oligomycin (5 μg/ml, Sigma-Aldrich) for 24 h. For quantification of colony-forming units (CFUs), the infected cells were lysed in distilled water and plated on 7H10 agar plates supplemented with OADC.

His-tagged TRAIL and FasL were produced and used as described previously.^{52 (link)} Staurosporine, glycerol, nonidet P-40 (NP40) and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich (Lyon, France). Bax channel blocker was from Santa Cruz Biotechnology (Tebu-bio, Le Perray en Yvelines, France). MG-132 (Cat# 1748) was from Tocis (Bristol, United Kingdom). The pan-caspase inhibitor z-VAD-fmk and cycloheximide (Cat# ALX-380-269) were from Enzo Life Science (Villeurbanne, France).

Western blot analysis was performed with the following antibodies: H86 for proinsulin (Santa Cruz, cat. # sc-9168), C4 for beta-actin (Millipore, lot. #LV1728681), H68.4 for transferrin-receptor (Invitrogen, cat. # 13–6800), mouse HRD1 (Cell Signaling, #12925), D8Z4G for UBE2g2 (Cell Signaling, #63182), and HCA2 and W6/32 against HLA. Secondary antibodies used were goat α-mouse IgG(H+L)-HRP (no. 170–6516, Bio-Rad) and goat α-rabbit IgG(H+L)-HRP (no. 4030–05, Southern Biotech). Cycloheximide was from Enzo Life Sciences.

α-Amanitin, cycloheximide, J-8, and cantharidic acid were purchased from Enzo Life Sciences (USA). Triptolide was purchased from MedChem Express (USA). 3,4-Methylenedioxy-β-nitrostyrene (MNS), 2-aminoethyl diphenylborinate (2-APB), and okadaic acid were purchased from Santa Cruz Biotechnologies (USA). The 81 chemicals used for the screening were provided by the Drug Discovery Initiative, The University of Tokyo (Japan) and are listed in S1 Table. All chemicals were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical, Japan; special grade) as a stock solution and stored at -20°C. Chemical solutions at the appropriate concentrations were prepared by diluting stock solutions in sterilized Milli-Q (stMQ) water just prior to chemical treatment. The final concentration of DMSO in all chemical solutions was adjusted to 1%.

Mammalian cells used in this study, including HEK293, HEK293-pre1-HTBH, HEK293-trex-htau40 and C2C12 cells, were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 units per ml penicillin/streptomycin. Cells were maintained in a humidified incubator with 5% CO₂ at 37 °C. For transfection, cells were treated with 1–2 μg of total plasmid DNA in a 6-well culture plate (>95% confluent or at a density of 10⁶ cells per well) for 36–48 h using Lipofectamine 3000 (Invitrogen). Cell lysates were prepared in RIPA buffer 36–48 h post transfection and were used for immunoblotting. For chase analysis, wild type and α3ΔN cells were treated with 75 μg ml⁻¹ cycloheximide (Enzo Life Sciences) and samples were isolated at chase times 0, 20, 40 and 60 min after 4 h transient MG132 (AG Scientific, San Diego, CA, USA) treatment and vigorous washing with phosphate-buffered saline (PBS). For stable cell line maintenance, transfected cells were cultured with DMEM medium containing 600 mg ml⁻¹ G418 (Santa Cruz) and 10% FBS. Fluorescence images were obtained after cells were extensively rinsed three times with PBS.