6 formylindolo 3 2 b carbazole ficz

Human umbilical cord blood (HUCB) was provided by Pusan National University Yangsan Hospital (PNUYH, IRB No. 05-2017-053), and mononuclear cells (MNCs) were isolated from HUCB using density gradient centrifugation with Ficoll (GE Healthcare, Buckinghamshire, UK). The freshly isolated MNCs were seeded in dishes coated with 1% gelatin (Sigma-Aldrich, St. Louis, MO, USA) and cultured in endothelial growth medium-2 (EGM-2; Lonza, Walkersville, MD, USA) consisting of endothelial cell basal medium (EBM-2), 5% fetal bovine serum (FBS), 1% penicillin-streptomycin, human basic fibroblast growth factor (bFGF), human vascular endothelial growth factor, human insulin-like growth factor-1 (IGF-1), ascorbic acid, human epidermal growth factor (EGF), and gentamicin sulfate-amphotericin (GA-1000). Cells were incubated at 37 °C in 5% CO₂. After 5 days, nonadherent cells were discarded, and the attached cells were cultured continuously. The cultures were maintained to allow the development of spindle-shaped colonies. EGM-2 was changed daily, and the colonies were replated and cultured further. To compare young and senescent EPCs, young EPCs were used at passages ≤ 8, and senescent EPCs were used at passages ≥ 14 in subsequent experiments. 6-Formylindolo 3 2-b carbazole (FICZ) was from Sigma-Aldrich.

RAW 264.7 cells, abelson murine leukaemia virus‐induced/immortalized male BALB/c mouse monocyte/macrophage cell line was purchased from American Type Culture Collection (ATCC). DMEM medium (Gibco) supplemented with 10% FBS were the main condition to culture cells. 6‐formylindolo[3,2‐b] carbazole (FICZ) (Sigma–Aldrich) or 2‐methyl‐2H‐pyrazole‐3‐carboxylic acid (2‐methyl‐4‐o‐tolylazo‐phenyl)‐amide (CH‐223191) (Selleck Chemicals LLC) were used to pretreat with RAW 264.7 cells for 12 and 24 h, respectively.

A549 cells were cultured in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO₂. PM_2.5 suspension was applied to A549 cells at a final concentration of 2 µg/mL for four days. BaP (Tokyo Chemical Industry, Tokyo, Japan) and 6-Formylindolo[3,2-b] carbazole (FICZ, Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 mM concentration. A549 cells were seeded into 6-well plates at 5×10⁴ cells concentration and treated with BaP or FICZ at the indicated concentrations and times. Control cells were treated with DMSO alone. For H₂O₂ stimulation, A549 cells were seeded into 6- or 12-well plates at 2×10⁵ or 5×10⁵ cells, respectively, and treated with 10 µM H₂O₂ (Nacalai Tesque, Tokyo, Japan) for 6 h. N-acetyl-l-cysteine (NAC, FUJIFILM Wako Chemicals, Tokyo, Japan) was reapplied to A549 cells at a final concentration of 1 mM for 1 h with 2 µM BaP.