K sfm

Normal human prostate epithelial cell line, RWPE-1, was obtained from the American Type Culture Collection (ATCC). Human prostate cancer cell line, 22Rv1, was provided by Dr. E. Diamandis (Mount Sinai Hospital). RWPE-1 cells were cultured with Keratinocyte serum-free medium (K-SFM) (Invitrogen) supplemented with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF). 22Rv1 cells were cultured with RPMI 1640 (Mount Sinai Hospital) with 10 % fetal bovine serum (FBS). All cells were cultured as a monolayer and maintained in a humidified incubator at 37 °C with 5 % CO₂. Genomic DNA was extracted from cells after trypsinization, using the QIAamp DNA Mini Kit (Qiagen) following the protocol provided.

The prostate cancer cell lines, LNCaP, PC3, and DU145 were grown in RPMI, whereas VCaP cells were grown in F12:DMEM and PNT2 in defined KSFM (Invitrogen, USA). All cells were incubated at 37 °C in 5% CO₂, and supplemented with 1% v/v penicillin, streptomycin, glutamine (PSG) and 10% v/v fetal calf serum (FCS).

Hydroxysafflor yellow A (98.05%, HY-N0567, MedChemExpree, USA), CsA (99.85%, HY-B0579, MedChemExpree, USA), N-acetyl-L-cysteine (NAC, A7250, Sigma-Aldrich, USA). Cell line’s basal medium and nonkeratinocyte culture medium (K-SFM, 17005-042, Invitrogen, Thermo, MA, USA). Radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (P0013B, Beyotime, Shanghai, China), rabbit antihuman Bax primary antibody (5023, Cell Signaling Technology, Inc. USA), and horseradish oxidase-linked goat antirabbit secondary antibody (7074, Cell Signaling Technology, Inc. USA). Super Signal Enhanced Chemiluminescence Kit (34094, Thermo, USA). The ones bought from Abcam (Cambridge, UK) are Rabbit antihuman Bcl-2 antibody (ab194583), ?-actin primary antibody (ab227387), anti-nuclear factor-?B (anti-NF-κB) p65 antibody (cab16502), and Schiff staining kit (ab150680). Superoxide dismutase (A001-3), malondialdehyde (A003-1), glutathione peroxidase (A005), ROS (E004), and peroxide hydrogenase (A007-1-1) detection kits were purchased from NJJC Bio (Nanjing, China). Hematoxylin and eosin (H&E) staining kit (C0105, Beyotime Biotechnology (Shanghai, China). Other reagents are of analytical grade.

PZ-HPV-7 cell line is a normal human prostate epithelial cell line acquired from the American Type Tissue Culture Collection (ATCC, Rockville, MA, USA). Cells were maintained in Keratinocyte-serum-free medium (K-SFM) supplemented with bovine pituitary extract (50 μg/ml) and epidermal growth factor (5 ng/ml) (Gibco/Invitrogen, Grand Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO₂. The medium was replaced every 2 or 3 days.

Human oral cancer cell line OC3-IV2 cells have been described [9 (link),25 (link),26 (link)] and were cultured in a 1:1 ratio of Dulbecco’s modified Eagle medium (DMEM; Invitrogen) and keratinocyte serum-free medium (KSFM; Invitrogen) containing 10% (v/v) fetal bovine serum (Invitrogen), 1% (v/v) l-glutamine (Invitrogen), and 1% (v/v) antibiotic-antimycotic (Invitrogen). Human primary glioblastoma cell line U87 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) Minimum Essential Medium (MEM) non-essential amino acids solution (Invitrogen), and 1% (v/v) penicillin/streptomycin (Invitrogen). Human prostate cancer cell line PC3 cells, human cervical cancer cell line HeLa cells, and 293T cells were obtained from the ATCC and maintained in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) l-glutamine, and 1% (v/v) antibiotic-antimycotic. Human non-small cell lung carcinoma cell line H1299 cells were obtained from the ATCC and were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) containing 10% (v/v) fetal bovine serum, 1% (v/v) l-glutamine, and 1% (v/v) antibiotic-antimycotic. Cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C, and the culture medium was replaced every 2 days.