Dfo d9533

Human neutrophils were isolated from fresh heparinized blood by density centrifugation at 500 x g using Polymorphprep™ (Axis-Shield PoC). Neutrophils have been seeded on cover slides covered with 0.01% Poly-L-lysine (# P4707, Sigma–Aldrich).
A total of 5×10⁵ cells in 250 μl RPMI 1640 (# E15-848, phenol red free, PAA) were seeded per well in a 24-well-plate. The cells were either stimulated with 25 nM PMA (Sigma–Aldrich), or 300 μM DFO (D9533, Sigma–Aldrich) for 3 h at 37°C with 5% CO₂. For some experiments, divalent or trivalent iron ions (250 μM) were additionally added. After incubation, the cells were fixed by adding PFA to each well at a final concentration of 4% for 15 min at room temperature and kept at 4°C until subsequent immunostaining.

The following antibodies were used in this study at the indicated dilution for western blot (WB) analysis, IP, IHC, and immunofluorescence (IF): Nedd4 (PA5-17463, Thermo, 1:1000 for WB, 1:100 for IP, 1:100 for IHC), VDAC1 (MABN504, Merck, 1:1000 for WB), VDAC2 (ab37985, Abcam, 1:1000 for WB), VDAC3 (ab130561, Abcam, 1:1000 for WB, 1:100 for IHC), FOXM1 (702664, Thermo, 1:1000 for WB), p-ERK (4370, Cell Signaling, 1:1000 for WB), ERK (4695, Cell Signaling, 1:1000 for WB), 4HNE (ab46545, Abcam, 1:100 for IHC), Flag (F3165, clone M2; Sigma, 1:1000 for WB, 1:100 for IP), HA (H6533, Sigma, 1:1000 for WB), GST (PA1982A, Thermo, 1:1000 for WB), Myc (AH00052, Thermo, 1:1000 for WB, 1:100 for IP), and Actin (PA116889, Thermo, 1:1000 for WB). Horseradish peroxidase (HRP)-labeled or fluorescently labeled secondary antibody conjugates were purchased from Molecular Probes (Thermo). Purified rabbit IgG was purchased from Pierce. Erastin (E7781), Ferrostatin-1 (S7243), Liproxstatin-1 (S7699), EUK134 (S4261), and Tempol (S2910) were obtained from Selleck (Houston, TX, USA). DFO (D9533) and CPX (SML2011) were obtained from Sigma (St. Louis, MO).

Cadmium chloride (CdCl₂, 202908), NAC (30498), and DFO (D9533) were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 12800-017), fetal bovine serum (FBS, Gibco, 10437-028), Trizol (Ambion, 15596026), and trypsin-EDTA (25200072) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The ROS detection kit (S0033S), Mito-Tracker Red fluorescent probe (C1035), and Bicinchoninic acid (BCA) protein assay kit (P0012) were from Beyotime Biotechnology Co., Ltd. (Beyotime, Shanghai, China). The MDA (A003-1-2) and GSH detection kits (A006-2-1) were purchased from Nanjing Jiancheng Bio-Engineering Institute Co., Ltd. (Nanjing, China). The Cell Counting Kit-8 (CCK-8, A311-01) was purchased from Vazyme Biotechnology Co., Ltd. (Vazyme, Nanjing, China). The PrimeScrip^TM RT Reagent Kit (RR037A) and SYBR Green™ Premix Ex Taq™ (RR390A) were obtained from Takara Biomedical Technology Co., Ltd. (Takara, Beijing, China). The RIPA lysate (20101ES60) was obtained from New Cell & Molecule Biotechnology Co., Ltd. (NCM, Suzhou, China). The Fer-1 (HY-100579) and Erastin (HY-15763) were purchased from MedChemExpress LLC (MCE, Princeton, NJ, USA). All chemicals were of the highest purity grade available.