MCA205 or GL261 cells were seeded at 105 cells/well in a 96-well plate. After incubation overnight, the cells were stained with 3.3 µM Sytox Green nucleic acid stain (molecular probes) and stimulated with 2.5 µM RAS-selective lethal 3 (RSL3) (Sigma Aldrich) for different durations (1, 3, 6 and 24 hours). Cell death was analyzed as described in Demuynck et al.36 (link) Fluorescence was measured on a Tecan Spark 20M multimode microplate reader.
The following inhibitors were used to block different cell death modalities36 37 (link): the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk, 25 µM, apoptosis, Bachem), the inhibitor of ROS and lipid peroxidation ferrostatin-1 (Fer-1, 1 µM, ferroptosis, Sigma Aldrich), the iron chelator deferoxamine (DFO, 10 µM, ferroptosis, Sigma Aldrich), the nonspecific lipophilic antioxidant α-tocopherol (α-toc, 100 µM, ferroptosis, Sigma Aldrich) and the RIPK-1 inhibitor necrostatin-1s (Nec-1s, 20 µM, necroptosis, Abcam). The cell death inhibitors were added 1 hour before RSL3 stimulation.
For recovery experiments on both MCA205 and GL261 cells, after treatment for 1, 3 or 6 hours, RSL3-conditioned medium (2.5 µM RSL3) was replaced with drug-free medium; cell death was determined 24 hours after RSL3 stimulation as described above.
The following inhibitors were used to block different cell death modalities36 37 (link): the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk, 25 µM, apoptosis, Bachem), the inhibitor of ROS and lipid peroxidation ferrostatin-1 (Fer-1, 1 µM, ferroptosis, Sigma Aldrich), the iron chelator deferoxamine (DFO, 10 µM, ferroptosis, Sigma Aldrich), the nonspecific lipophilic antioxidant α-tocopherol (α-toc, 100 µM, ferroptosis, Sigma Aldrich) and the RIPK-1 inhibitor necrostatin-1s (Nec-1s, 20 µM, necroptosis, Abcam). The cell death inhibitors were added 1 hour before RSL3 stimulation.
For recovery experiments on both MCA205 and GL261 cells, after treatment for 1, 3 or 6 hours, RSL3-conditioned medium (2.5 µM RSL3) was replaced with drug-free medium; cell death was determined 24 hours after RSL3 stimulation as described above.
