F12k medium

PHC was purchased from Lisite Corporation (Chengdu, China). The t-BHP, brusatol (Nrf2 inhibitor) and CC (AMPK inhibitor) were purchased from Invitrogen-Gibco (Grand Island, NY, USA). F-12K medium, fetal bovine serum, dimethyl sulfoxide, penicillin and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Malondialdehyde, myeloperoxidase, SOD and glutathione assay kits were supplied by Biolegend (San Diego, CA, USA). Rat IL-6, IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Cell Signaling (Boston, MA, USA). Antibodies against IL-1β, caspase-1, ASC, NLRP3, IκBα, p-IκBα, GSK3β, p-GSK3β, AMPK, p-AMPK, GCLC, GCLM, NQO1, HO-1, Nrf2, GAPDH and histone H3.1 were purchased from Protein-Tech (Boston, MA, USA). Unless otherwise specified, all other reagents were purchased from the Jiancheng Bioengineering Institute of Nanjing.

The rat pheochromocytoma (PC-12) cell line was purchased from ATCC (American Type Culture Collection). Kaighn’s Modification of Ham’s F-12 Medium (F-12 K medium), NGF-7S from murine submaxillary gland, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), tropomyosin receptor kinase (Trk) receptor inhibitor (K252a), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) inhibitors (U0126, PD98059), phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) inhibitor (LY294002), were purchased from Sigma Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and horse serum (HS) were purchased from PAA Laboratories (Cölbe, Germany). ChemiKine™ nerve growth factor sandwich enzyme-linked immunosorbent assay (ELISA) kit was purchased from Chemicon® International, Inc. (USA). All chemicals used were of analytical grade.

The immortalized human normal bladder epithelial cell line SV-HUC-1 and human BCa cell lines UMUC3, 5637, T24, and EJ were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). UMUC3, 5637, T24, and EJ cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, United States) and SV-HUC-1 cells were maintained in F12K medium (Sigma-Aldrich; Merck KGaA, Germany). All cell cultures were supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, United States) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, United States) and cultured at 37°C in a humidified incubator containing 5% CO₂.

A mouse normal hepatocyte cell line, FL83B, was maintained in F-12K medium (Sigma-Aldrich) supplemented with 10% FBS (Gibco, Charlemont Terrace, Dublin, Ireland), 0.15% sodium bicarbonate (Sigma-Aldrich), 100 mM nonessential amino acids (NEAAs, Gibco), 100 unit/mL penicillin, and 100 µg/mL streptomycin (Gibco). Cultured cells were incubated at 37 °C with 5% CO₂. Cells were seeded in a 6-well plate at a density of 5 × 10⁵ cells/well. After the cells grew to 80% confluence, the PmAlb-BDD-FVIII-pCMV-EGFP plasmid was transfected with Lipofectamine 2000 (Invitrogen) as described previously [37 (link)].