Mesenchymal-like ovarian cancer cells are more susceptible to ferroptosis than epithelial-like ones.

a. Overnight-starved cells were plated into Matrigel-containing chambers and allowed to invade for 24 h. After removing contents in upper chambers, cells on the undersurface of chambers were stained with crystal violet and quantified using Image J. b. Overnight-cultured cells were lysed and cell lysates were subjected immunoblotting with antibodies against vimentin, E-cadherin or GAPDH. c. Cells were treated with varying concentration of ML162 for 24 h, followed by an MTT assay to determine cell viability. Data are means ± SEM. n = 3. d. Cells were treated with varying concentration of erastin for 24 h, followed by an MTT assay to determine cell viability. Data are means ± SEM. e. OCC1, OVCAR8 and ES2 cells were treated with vehicle, ML162 or erastin in the absence or presence of 10µM ferrostatin-1 for 24 h, followed by an MTT assay to analyze cell viability. % of cell viability was calculated as [(Control – Treatment)/Control] x 100. Control: vehicle-treated; Treatment: ML162 or erastin with or without ferrostatin-1. Data are means ± SEM. ****, P < 0.0001. f. Cell lysates of overnight-cultured cells were subjected to immunoblotting with antibodies against SLC7A11, GPX4 or GAPDH.

Ferroptosis occurs in a similar mechanism of pyroptosis in ovarian cancer cells.

a. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 200nM ML162 in the absence or presence of YVAD, DEVD, necrostatin or chloroquine for 24 h, followed by an MTT assay to analyze cell viability. % of cell viability was calculated as [(Control – Treatment)/Control] x 100. Control: vehicle-treated; Treatment: ML162 with or without PCD individual inhibitors. Data are means ± SEM. ****, P < 0.0001. b. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 3µM erastin in the absence or presence of individual PCD inhibitors for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. c. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 200nM ML162 in the absence or presence of disulfiram for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. d. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 3µM erastin in the absence or presence of disulfiram for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. e. OCC1, OVCAR8 and ES2 cells were treated with vehicle, 200nM ML162 (left panel) or 3µM erastin (right panel) in the absence or presence of YVAD or disulfiram for 24 h and conditioned media were then collected to measure LDH activity. Data are means ± SEM. n = 3. ****, P < 0.0001. f. OCC1, OVCAR8 or ES2 cells were treated with vehicle, 200nM ML162 (left panel) or 3µM erastin (right panel) for 24 h and conditioned media were then collected to measure the level of IL-1β. Data are means ± SEM. ****, P < 0.0001.

The mechanism of canonical inflammasome pyroptosis is not involved in ferroptosis.

a. OCC1, OVCAR8 and ES2 cells were first transfected with control or ASC siRNA pool for 2 days and then treated with 200nM ML162 for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. b. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 200nM ML162 in the absence or presence of ADS032 or YVAD for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. c. OCC1, OVCAR8 and ES2 cells were treated with vehicle or 200nM ML162 in the absence or presence of MCC950 for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. d. OCC1, OVCAR8 and ES2 cells were treated with varying concentrations of ML162 or erastin for 24 h and then lysed for immunoblotting to detect caspase-1 (CASP1) with anti-CASP1 antibody. Membranes were stained with Ponceau S to ensure equal loading. e. OCC1, OVCAR8 and ES2 cells were first transfected with control or CASP1 siRNA pool for 4 days and then treated with 200nM ML162 or 3µM erastin for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM.

Caspase-5 is activated in cells exposed to ferroptosis inducers and its presence is necessary for ferroptosis.

a. OCC1, OVCAR8 and ES2 cells were treated with varying concentrations of ML162 or erastin for 24 h and then lysed for immunoblotting to detect caspase-4 (CASP4) or CASP5 (CASP5) with the respective antibodies. Membranes were stained with Ponceau S to ensure equal loading. b. OCC1, OVCAR8 and ES2 cells transfected with control or CASP4 siRNA pool were treated with 200nM ML162 or 3µM erastin for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. c. OCC1, OVCAR8 and ES2 cells transfected with control or CASP5 siRNA pool were treated with 200nM ML162 or 3µM erastin for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001.

Caspase 5 activity is critical for ferroptosis.

a. OCC1 and OVCAR8 cells were treated with varying concentrations of ML162 for 24 h followed by analyzing caspase-5 activity. Experiments were performed three times, and a representative result was shown here. Data are means ± SD. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. b. CASP5-KO clones 111 and 69 of OCC1, CASP5-KO clones 63 and 3 of OVCAR8 cells were treated with 200nM ML162 for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. c. OCC1 CASP5-knockout clone 111 and OVCAR8 CASP5-knockout clone 63 were lentivirally transduced with wild-type or caspase-dead CASP5 mutant (CASP5/C328A) for 3 days and then treated with 200nM ML162, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001.

GSDME is cleaved by caspase-5 to be engaged in ferroptosis.

a. OCC1, OVCAR8 and ES2 cells were treated with varying concentrations of ML162 or erastin for 24 h and then lysed for immunoblotting to detect GSDME with the respective antibodies. Membranes were stained with Ponceau S to ensure equal loading. b. OCC1, OVCAR8 and ES2 cells transfected with control or GSDME siRNA pool were treated with 200nM ML162 or 3µM erastin for 24 h, followed by an MTT assay to analyze cell viability. Data are means ± SEM. ****, P < 0.0001. c. 5.0µg of recombinant wild-type GSDME or GSDME mutant (D270A) was incubated with 1U of active caspase-5 in caspase reaction buffer at 37°C for 1 h. After termination the reactions, samples were resolved on 12% SDS–PAGE gels, stained with Coomassie and imaged. Red star denotes the band of cleaved GSDME (CL-GSDME). Red arrow points the position of full-length GSDME (FL-GSDME).