Abt 737

Bax^−/−Bak^−/− MEFs and those reconstituted with the indicated BAX proteins were cultured as described above, plated in 96-well plates (1.5 × 10³ cells per well), and allowed to adhere for 24 h. The cells were then treated with 10 μM ABT-737 (Cayman Chemical) and the indicated concentration of S63845 (SelleckChem), or vehicle (0.05% and 0.1% DMSO, respectively) for 24 h. Cell viability was measured by CellTiter-Glo assay (Promega) with luminescence read on a Spectramax M5 microplate reader equipped with SoftMax Pro 7.0.2 software.

TRAIL was commercially supplied (Enzo Life Sciences, Farmingdale, NY, USA) or produced from 293T cells as described.^{45 (link)} Briefly, the extracellular portion of human TRAIL (amino acids 95–281) was cloned into pBabe retroviral expression vectors to produce functionally active TRAIL,^{46 (link)} which was quantified by TRAIL ELISA according to the manufacturer’s instructions (Abcam, Cambridge, UK). Caspase inhibitors (BD Pharmingen, San Diego, CA, USA) were as follows: Z-IETD-FMK (caspase-8 inhibitor), Z-VAD-FMK (general caspase inhibitor), Z-FA-FMK (negative control), Z-LEHD-FMK (caspase-9 inhibitor) and Ac-DEVD-CHO (caspase-3/7 inhibitor). Bcl-2, Bcl-xL inhibitors ABT-263 and ABT-737 were purchased from Cayman Chemicals (Ann Arbor, MI, USA)

CL316243, isoproterenol, resveratrol, rosiglitazone, amlexanox, zardaverine, H89, IBMX, and thiazolyl blue tetrazolium blue (MTT) were from Sigma. [1-¹⁴C] palmitic acid was from Moravek Biochemicals. 2′3′-cGAMP and 2′3′-cGAMP control were from Invivogen. ABT-737, Q-VD-OPh and nigericin were from Cayman Chemical. Digitonin was from EMD Millipore. Antibodies to UCP1 (Cat# Ab23841), C/EBPβ (Cat# Ab32358), ChREBP (Cat #157153), Prdm16 (Cat# ab106410), and ERP57 (Cat# Ab10287) were obtained from Abcam; antibodies to cGAS (Cat# 31659), p-TBK1 (Cat# 5483), TBK1 (Cat# 3013), p-IRF3 (Cat# 4947), IRF3 (Cat# 4302), STING (Cat# 13647), PKA Substrates phosphorylation (Cat# 9621), p-HSL (Cat# 4139), HSL (Cat# 4107), PPARγ (Cat# 2443), ATGL (Cat# 2439), FASN (Cat# 3180), p-ACC (Cat# 3661), ACC (Cat# 3662), and SCD1 (Cat# 2794) were all from Cell Signaling; an antibody to PGC1α (Cat# ST1204) was from EMD Millipore; an antibody to TNFα (Cat# CG1601) was from Cell Applications, Inc; an antibody to Complex IV (Cat# A6403) was from Molecular Probes; Lamin A antibody (Cat# 3267-100) was from BioVision; antibodies to beta-tubulin and myc were made from monoclonal antibody-producing cell lines obtained from ATCC; A polyclonal anti-DsbA-L antibody is homemade from rabbit and also available in EMD Millipore (Cat# ABS1644).

Mice were subcutaneously injected twice (on days P10 and P12) with ABT-737 (Cayman Chemical) or vehicle control at a dose of 75 mg kg⁻¹.

Caski and SiHa cervical cancer cell lines were obtained from the American Type Tissue Culture Collection. The Caski cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.2% sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The SiHa cells were cultured in Dulbecco’s Modified Eagle Medium with 10% FBS, 5.5% sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. As₂O₃, 3-methyladenine (3-MA), and chloroquine were purchased from Sigma (St. Louis, MO, USA). ABT-737 was obtained from Cayman (Ann Arbor, MI, USA).
MTT (3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) assay (MTT assay). The Caski and SiHa cells (5 × 10³ cells/well) were seeded into a 96-well plate with a 100 μL culture medium. The medium was removed after treatment for 48 h, and 100 μL of the medium containing 0.5 mg/mL MTT (Sigma, M 2128) was added. MTT assay was performed as previously described [19 (link)]. The combination index was calculated using the software Compusyn 1.0. [20 (link)].