Sb239063

The human breast carcinoma cell lines MDA-MB-231 and MCF-7 were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultivated in RPMI-1640 (Gibco/BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco/BRL), 2 mg/ml sodium bicarbonate (Gibco/BRL), 100 U/ml penicillin (Gibco/BRL), and 100 μg/ml streptomycin (Gibco/BRL).
Cells were seeded in 96-well plates (1.5×10⁴ cells/well) and incubated at 37°C in a 5% CO₂ atmosphere. To determine the IC₅₀ of FK-3000, MDA-MB-231 and MCF-7 cells were treated with 0.1% dimethyl sulfoxide (DMSO; vehicle only control) (Sigma-Aldrich Co.) or FK-3000 (0–5 μg/ml) 24 h after seeding. Cell proliferation was analyzed after 24 and 48 h using the cell counting kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer’s instructions.
To evaluate cell viability, cells were treated with 0.1% DMSO, the 48 h IC₅₀ of FK-3000 (0.52 μg/ml for MDA-MB-231 cells; 0.77 μg/ml for MCF-7 cells), 5.0 μM trans-1-(4-hydroxy-cyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)-imidazole (p38 MAPK inhibitor SB 239063, Sigma-Aldrich Co.), or cotreated with the 48 h IC₅₀ of FK-3000 and 5.0 μM SB 239063. Cell viability was evaluated after 48 h using the cell counting kit-8 (Dojindo Molecular Technologies). All experiments were performed in quadruplicate on different days.

The rat kidney proximal tubular epithelial (NRK-52E) cells were purchased from SIBS (Shanghai Institutes for Biological Sciences). NRK-52E cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) under 5% CO₂/95% atmosphere at 37 °C incubator, and serial sub-cultivation in T-25 flasks. DMEM, FBS, penicillin and streptomycin were bought from Gibco (USA), NRK-52E cells were cultured in 6- and 24-well plates. COM crystals were added at final different concentrations of 0, 36.5, 73.0, 109.5, 146, and 182.5 µg/cm², respectively. NRK-52E cells grew to 70–80% confluence in complete medium and the different concentrations of COM crystals were added, after incubated for 0, 12 and 24 h, the cells and medium were separately collected for further analysis. SB239063 (purchased from Sigma), an inhibitor of the activation of p38 MAPK, was prepared in PBS. SB239063 was added at final different concentrations of 20 mM and incubated with NRK-52E cells for 2 h and then the cells were collected for further analysis.

For in vitro drug screening, the SCADS inhibitor kits (kit 1, ver 3.3; kit 2, ver 2.0; kit 3, ver 1.6; kit 4, ver 2.3) were kindly provided by Molecular Profiling Committee, Grant-in-Aid for Scientific Research on Innovative Areas “Platform of Advanced Animal Model Support” from The Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI 16H06276). For the following experiment, dabrafenib (BRAF inhibitor), PD0325901 (MEK inhibitor) and SCH772984 (ERK inhibitor) were purchased from Selleck. LY3009120 (pan-RAF inhibitor) was purchased from Cayman Chemical. SB239063 (p38 inhibitor) and SP600125 (JNK inhibitor) were purchased from Sigma-Aldrich. These inhibitors were reconstituted in DMSO and stored at −20 °C or −80 °C.