Piperlongumine

Piperlongumine (>97%) was purchased from Sigma-Aldrich Chemical Co. (Schnelldorf, Germany), as dibasic sodium phosphate (>99%) reagents, anhydrous citric acid (>99%), and sodium chloride (>99%). Lyophilised Nucleotide Binding Domain of Heat Shock Protein 70 kDa (>97%) was purchased from GenScript. Methanol was purchased from Dynamics Química Contemporânea LTDA (Indaiatuba, SP, Brazil). All the materials purchased were used as supplied. Ultrapure water was prepared by a Millipore water purification system -Direct-Q UV-3(Merck KGaA, Darmstadt, Germany). Lyophilized NBD was reconstituted in a 50 mM phosphate buffer containing 150 mM sodium chloride, and the pH was adjusted to 7.4 with anhydrous citric acid. Stock solutions of PPL were prepared in methanol. The concentrations of PPL and NBD solutions were determined by UV-Vis experiments performed on Biospectro spectrophotometer (Biospectro, Curitiba, PR, Brazil), using the extinction coefficient of 18700 M⁻¹·cm⁻¹ at 326 nm for PPL and 20,525 M⁻¹·cm⁻¹ at 280 nm for NBD.

Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in a humidified atmosphere containing 5% CO2 at 37°C in Endothelial Growth Medium-2 (EGM2, Lonza, Switzerland). For hypoxia induction, cells were incubated in a hypoxia chamber with a mixture of 1% O2, 5% CO2, and 94% N2. For TNF-α stimulation, 10 ng/ml of recombinant human TNF-α (BD Biosciences, USA) was added into the HUVECs medium. Piperlongumine (Sigma-Aldrich, USA) was dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) before usage. The culture medium was replaced every 1-2 days, and cells at 85-90% confluence were passaged at a ratio of 1:2 confluence.

The K562 human leukemia cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and the doxorubicin-resistant K562/A02 cell line was purchased from the Tianjin Institute of Hematology (Tianjin, China). The cells were cultured at 37°C with 5% CO₂ in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA). Piperlongumine (Sigma Aldrich) was dissolved in DMSO to prepare a stock solution, which was further diluted to the experimental concentrations with serum-free culture medium and filtered immediately.

Auranofin (Santa Cruz, CA) and Piperlongumine (Sigma, St. Louis, MO) were suspended in dimethyl sulfoxide and stored in volumes of 1 mL at −20°C. Human gastric cancer cell lines SGC-7901, BGC-823 and KATO III were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetal bovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37°C. Antibodies including anti-p-PERK, anti-Bcl-2, anti-Bax, anti-cleaved PARP, anti-caspase-3 p30/17, anti-GAPDH, goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-CHOP, anti-ATF4, anti-p-eIF2α, anti-Cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ).

Piperlongumine (PL), N-acetyl-L-cysteine (NAC), Necrostatin-1 (Nec1), Ferrostatin-1, Liproxstatin, Glutathione (GSH), Ciclopirox (CPX) were purchased from Sigma-Aldrich (Sigma, St. Louis, USA), zVAD was from Promega (Promega, Madison, WI, USA).
The primary antibodies and their dilutions used for western blotting experiments were as followed, beta-actin (A5441, 1:1000, Sigma, St. Louis, USA), PARP (sc-7150, 1:1000, Santa Cruz Biotechnology, Santa Cruz, USA), cleaved-caspase 3 (9661, 1:1000, Cell Signaling, Danvers), P-JNK (4668, 1:1000, Cell Signaling, Danvers), JNK (sc-571, 1:1000, Santa Cruz Biotechnology, Santa Cruz, USA), P-c-Jun (9261, 1:1000, Cell Signaling, Danvers), OPA1 (BD Bioscience 612606), PRC1 (1:1000, Protein Tech Group, Chicago, USA), FOXM1 (sc-376471, 1:1000, Santa Cruz Biotechnology, Santa Cruz, USA), GPX4 (ab125066, 1:1000, Abcam, Cambridge, UK).
The secondary antibodies used for Western blotting experiments were as followed: ECL anti-rabbit IgG horseradish peroxidase linked, ECL anti-mouse IgG horseradish peroxidase linked (Amersham Biosciences, Otelfingen, Switzerland).

Piperlongumine (PL), N-acetyl-L-cysteine (NAC), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 and SP600125 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). DMEM and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Antibodies against NFκB p65, IκBα, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), and the antibodies against p-p38, total p38, p-JNK, and total-JNK were from Cell Signaling Technology.