Attune software v2

Lipid peroxidation was measured by flow cytometry with the use of BODIPY 581/591 C11 (Thermo Fisher Scientific). Cells were incubated with drug under normoxic or hypoxic conditions for 18 h. After the addition of BODIPY 581/591 C11 to a final concentration of 5 μM, the cells were incubated for an additional 30 min, isolated by exposure to trypsin, stained for 5 min with PI in DMEM for dead cell exclusion, and then analyzed with an Attune flow cytometer (Thermo Fisher Scientific Inc.) with excitation at 488 nm and a 530/30-nm emission filter to detect oxidized forms of the probe. MFI was calculated with Attune software v2.1.0 (Thermo Fisher Scientific Inc.).

Mitochondrial superoxide production was measured by flow cytometry with the use of MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific Inc.). Cells were incubated with drug under normoxic or hypoxic conditions for 12 h. After the addition of MitoSOX Red to a final concentration of 5 μM, the cells were incubated for an additional 10 min and then analyzed with an Attune flow cytometer (Thermo Fisher Scientific Inc.). Mean fluorescence intensity (MFI) was calculated with Attune software v2.1.0 (Thermo Fisher Scientific Inc.).