Mcf 7

MCF-7 and CAMA-1 breast cancer cells (from American Type Culture Collection) were cultured in cell culture dishes (Falcon) with RPMI-1640 medium (Corning) supplemented with fetal bovine serum (FBS) (10%, Biosera), sodium pyruvate (1 mM, Corning), penicillin (100 IU/mL, Corning), and streptomycin (100 μg/mL, Corning). The cell line identities were confirmed with a Multiplex Human Cell Line Authentication Test (Multiplexion) and tests for Mycoplasma infection were done every other month (Eurofins). When indicated, cells were treated with LCL161 (MedChem Express) and/or TRAIL (Millipore) where 0.1% DMSO (Sigma-Aldrich) or ddH₂O was used as control. In addition, pre-treatment with inhibitors against caspases (zVAD-FMK, Enzo Life Sciences), caspase-8 (zIETD-FMK, MedChem Express), RIP1 kinase (Necrostatin-1, Sigma-Aldrich), and Janus kinases (Ruxolitinib, Selleckchem) was carried out when indicated.

The MCF-7 cell line (human breast cancer epithelial cells) was obtained from the National Centre for Cell Science, Pune, India. For the culture maintenance and in vitro cytotoxicity experiment, MCF-7 cells were cultured in a culture flask or on plates (Falcon, Sigma-Aldrich, Bengaluru, India) in DMEM, supplemented with 20% heat inactivated FBS, 1% penicillin, streptomycin, and amphotericin B in stable conditions of 37 °C and 5% CO₂ in a humidified atmosphere. A passage no higher than 20 was used (P18 in most of the experiments). The cells were frequently tested against mycoplasma according to the laboratory protocol [27 (link)].