Dulbecco modified eagle medium (dmem)

Human aortic endothelial cells (HAECs) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in DMEM (Gibco, Gaithersburg, USA) with 10% (v/v) fetal bovine serum (FBS, ExCell Bio, South America), 4.5 g/l glucose, 4.0 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in an incubator with 5% CO₂.
HAECs were divided into five groups: (1) control group (serum-free DMEM), (2) model group (0.4 mmol/l FeCl₃), (3) low-dose ALR-S group (0.1 mg/ml ALR-S), (4) middle-dose ALR-S group (0.2 mg/ml ALR-S), and (5) high-dose ALR-S group (0.4 mg/ml ALR-S). HAECs (1 × 10⁵) were seeded on six-well plates and pretreated at 90% confluence with different concentrations of ALR-S or vehicle for 4 h and then exposed to 0.4 mmol/l FeCl₃ for 2 h.
For ERK1/2 inhibition study, HAECs were divided into five groups: (1) control group (serum-free DMEM), (2) model group (0.4 mmol/l FeCl₃), (3) ALR-S (0.4 mg/ml ALR-S), and (4) U0126 (10 μmol/l U0126). HAECs (1 × 10⁵) were seeded on six-well plates and pretreated at 90% confluence with ALR-S or U0126 for 4 h and then stimulated with 0.4 mmol/l FeCl₃ for 2 h.

The human lung cancer cell lines A549, H358, and H460 and the lung epithelial cell line 16HBE were purchased from the Cell Bank at the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences and were cultured in 1640 medium supplemented with 10% foetal bovine serum and 1% double antibiotics, as instructed by their provider. HEK293T and HeLa cells were cultured in DMEM and 10% foetal bovine serum (ExCell Bio, Shanghai, China) supplemented with 1% double antibiotics. All cells were maintained at 37°C with 5% CO2. The cells were treated with Importazole (ab146155) [27 (link)], Leptomycin B (ethanol solution, ab120501) or Bufexamac (S3023, Selleck, Shanghai, China) for the indicated times.

LNCaP, DU145, 22Rv1, PC-3, and WPMY-1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM medium supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China). The cells were maintained in an incubator at 37 °C containing 5% CO₂. The androgen treatment assays were performed as described previously [31 (link)]. Briefly, LNCaP cells were starved for 3 days and then treated with DHT. The cells were collected and used in the following detection.