4.2. Treatment of HT-1080 and MIA PaCa-2 Cells for MTT Viability Assay, Flow Cytometry or GSH Measurement

The HT-1080 cells were seeded homogenously 24 h prior to treatment in either 96-, 24-, or 6-well plates (Thermo Scientific™ BioLite) at a seeding density of 1.5 × 10⁴, 1 × 10⁵, 5 × 10⁵ cells, respectively, in complete culture medium. MIA PaCa-2 cells were seeded homogenously 24 h prior to treatment in 96-well plates (Thermo Scientific™ BioLite) at a seeding density of 3 × 10⁴. After 24 h of incubation, the complete culture medium was renewed and supplemented with various compounds for treatment.

For ferroptosis induction, cells were treated with 0.05–5 μg/mL RSL-3 (MCE^®, Monmouth Junction, NJ, USA, HY-100218A, solved in DMSO) or 0.5–10 μM erastin (MCE^®, HY-15763, solved in DMSO). For inhibitor profile studies, the RSL-3 or erastin supplemented complete growth medium was further supplemented by one or two of the following: 5 μM final concentration of SP600125 (MCE^®, HY-12041, solved in DMSO) for HT-1080 cells, 10 μM final concentration for MIA PaCa-2 cells, 10 μM final concentration of JNK-IN-8 (MCE^®, HY-13319, solved in DMSO), 50 μM final concentration of Z-VAD-FMK (MCE^®, HY-16658B, solved in DMSO), 50 μM final concentration of necrostatin-1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-200142, solved in DMSO), 500 nM final concentration of liproxstatin-1 (MCE^®, HY-12726, solved in DMSO), 5 μM final concentration of ferrostatin-1 (MCE^®, HY-100579, solved in DMSO) for HT-1080 cells, 10 μM final concentration for MIA PaCa-2 cells.

For the measurement of apoptosis markers, camptothecin (Selleckchem, Houston, TX, USA, S1288, solved in DMSO) was used as a positive control in 5–10 μM final concentrations.

To investigate the effect of JNK inhibition on ascorbate induced cell death in HT-1080 cells, 100 mM ascorbic acid (Sigma-Aldrich^®, A4544) stock solution was prepared before each treatment in complete culture medium and the pH was adjusted to phenol red neutral with sodium hydroxide. Cells were treated with 0.1–5 mM ascorbate supplemented culture medium with or without further supplemented 5 μM final concentration of SP600125, 10 μM final concentration of JNK-IN-8, or 5 μM final concentration of ferrostatin-1.

For L-Buthionine-sulfoximine (BSO, Sigma-Aldrich^®, B2640) treatment, 100 mM BSO stock solution was prepared before each treatment in distilled water. The cells were seeded homogenously in 24-well plates in complete culture medium supplemented with BSO in 100 μM final concentration. After 24 h of incubation, the culture medium could be further supplemented with 5 μM final concentration of SP600125 or 10 μM final concentration of JNK-IN-8. After an additional 24 h of treatment time, cells were processed for either MTT assay or GSH measurement.

Solvent (DMSO) controls were used in all cases for inhibitor profile studies, max. DMSO content was 0.2 v/v%).