Caspase inhibitor z vad fmk

The full-length complementary DNA (cDNA) of GPX4 (human GPX4 GenBank accession no. NC_000019) was obtained from RiboBio Co., Ltd. (Guangzhou, China) and ligated into the NheI-XhoI site of the pcDNA3.1 vector (Life Technologies; Thermo Fisher Scientific, Inc.). The NheI and XhoI restriction enzymes and T4 ligase were purchased from Takara Bio (Heidelberg, Germany). A total of 0.8 µg of the plasmid or empty vector was mixed with 4 µl Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Inc.) in 0.5 ml Opti-MEM medium (Thermo Fisher Scientific, Inc.) and this was applied to cells for 4 h followed by medium-refresh. After 24 h, the culture medium was refreshed containing 500 µg/ml geneticin sulfate 418 (G418; Sigma-Aldrich; Merck KGaA) for antibiotic selection. Two weeks later, survival-transfected cells were collected and maintained in 250 µg/ml G418.
For the apoptosis assay, cells were pretreated with 20 µM caspase inhibitor Z-VAD-FMK (Promega Corporation, Madison, WI, USA), 1 µM Ferrostatin-1 (Ferr-1; Sigma-Aldrich; Merck KGaA), or 0.3 µM Necrostatin-1 (Nec-1; Sigma-Aldrich; Merck KGaA) for 12 h followed by co-incubation of H 2 O 2 or erastin, respectively. Twenty-four hours later, the cells were washed 3 times with PBS and harvested for further analysis.