Birinapant

HeLa (human cervical cancer cells), A375 (human malignant melanoma cells), Jurkat (human leukaemia T cells) and HEK-293-T (human embryonic kidney cells) were purchased from ATCC.
The DKO BAK/BAX MEFs (mouse embryonic fibroblasts) were kindly provided to us by Dr. Joe Opferman, St. Jude, Memphis, TN, USA, and by Dr. Reuven Stein, Tel-Aviv University, Israel.
MEFs cells, HeLa, A375 and HEK-293-T cells were grown in complete DMEM medium (1% sodium pyruvate, 1% l-glutamate, 1% Pen-strep and 10% fetal bovine/calf serum).
Jurkat and T47D (human metastatic ductal breast carcinoma cells) cells were grown in complete RPMI medium (1% sodium pyruvate, 1% l-glutamate, 1% Pen-strep and 10% heat-inactivated fetal bovine/calf serum).
184A1 (normal human breast epithelial cells) were grown in DMEM/F12 complete medium (1% sodium pyruvate, 1% l-glutamate, 1% Pen-strep, 5% donor horse serum, 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor, 0.5 mg/ml hydrocortisone, 10 µg/ml insulin).
All cell lines were checked for mycoplasma and kept under passage 10.
Staurosporine (STS) was purchased from Fermentek (cat#62996-74-1.5) and Birinapant from Biovision (cat#5297).

For cell death analyses, 1 × 10² cells were seeded in collagen-coated 96-well plates (Corning, BioCoat, 354407). Keratinocytes were pretreated for 30 min with Z-VAD-fmk (Enzo, ALX-260-020-M005, 20 μM) and Birinapant (BioVision, 2597) and stimulated by 20 ng ml⁻¹ TNF for 24 h in the presence of the dead stain Draq7 (Invitrogen, D15106, 0.3μM). Cell death assays were performed using the IncuCyte bioimaging platform (Essen); two to four images per well were captured, analysed and averaged. Cell death was measured by the incorporation of Draq7.

Doxycycline, Saquinavir, Azidodeoxythymidine (AZT), Nevirapine, and Raltegravir were purchased from Santa Cruz Biotechnology; human TNF-α was from Cell Signaling Technology; z-VAD-fmk was from R&D Systems; Birinapant was from BioVision; recombinant HIV-1 protease was from Prospec Tany TechnoGene; cOmplete Protease Inhibitor tablets were from Roche Diagnostics; all primers used for PCR and qRT-PCR were from IDT.