Z vad fmk z vad

The cell viability was measured using the CCK8 kit (MCE, HY-K0301) in the 96-well plate. The cells were seeded at 30-50% confluence (depends on the cell's growth rate), when the cells attached, firstly incubated with Ferrostatin-1 (Ferr-1) (Sigma, SML0583), Z-VAD-FMK (Z-VAD) (Selleck, S7023), Necrostain-1s(Nec-1s) (Selleck, S8641), Deferoxamine mesylate (DFO) (Selleck, S5742) or glycogen phosphorylase inhibitor (GPI) (Merck, 361515) for 12 h. Then co-treated with Erastin (Sigma, E7781) or tert-Butyl hydroperoxide (TBHP) (Sigma, 458139) for indicated time. Then all medium was removed, replaced with the fresh medium containing CCK8 reagent and incubated for 2-4 h, the plates were read by microplate reader at 450 nm.

eFT226 was synthesized by WuXi AppTec (Shanghai, China, purity >98%); ferrostatin-1 (Ferr-1), necrostatin-1 (Nec-1), and z-VAD-FMK (z-VAD) were obtained from SelleckChem (Houston, TX, USA); and other chemicals were obtained from Sigma-Aldrich (Burlington, MA, USA) unless specified elsewhere.

OR21 was synthesized by OHARA Pharmaceutical Co. AZA and DAC were purchased from Sigma-Aldrich. Ven and P62-mediated mitophagy inducer (PMI) were purchased from MedChemExpress. S63845, a selective myeloid cell leukemia-1 (MCL-1) inhibitor, and Z-VAD-FMK (Z-VAD) were purchased from Selleck Chemicals. Rapamycin (Rapa) was purchased from Tokyo Chemical Industry. All reagents were dissolved in DMSO and stored at −20°C.

The NSCLC cell lines (A549, H1299) purchased from American Type Culture Collection (ATCC) and the normal lung cells BEAS-2B, commercially obtained from the FuDan IBS cell center, were all maintained in complete Dulbecco's Modified Eagle' Medium (DMEM) (Gibco), supplemented with 10% FBS (Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin, at 37°C in a humidified incubator with 5% CO₂. The phenotype of cells used in the study were shown in Supplementary Figure S6. (−)-Guaiol, commercially obtained from Sigma, was diluted in methanol at a primary stock concentration 20 mM. 3MA, purchased from Sigma, was diluted in phosphate buffer saline (PBS, pH=8.0) at a stock concentration 0.5 M. MG132, a proteasome inhibitor [33 (link)], was diluted in DMSO at a final concentration 40 mM. The pan-caspase inhibitor Z-VAD-FMK (Z-VAD) was obtained from Selleck and diluted in DMSO at a stock concentration 5 mM. For the treatments, cells were treated with 150 μM (−)-Guaiol for 24 h in serum free medium, unless otherwise indicated. For the inhibition of autophagy or proteasome, 5 mM 3MA in serum free medium or 40 μM MG132 in complete medium were used after (−)-Guaiol treatment for another 6 h. For the caspase activity inhibition, 20 μM Z-VAD was incubated with (−)-Guaiol in serum free medium for 24 h.