Cercosporamide

HEK293T, MCF7 and HeLa cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 50 U of penicillin G/ml and 50 μg of streptomycin sulfate at 37 °C in 5% CO₂. HUVECs were purchased from Lonza and cultured in endothelial cell basal medium (EBM-2; Lonza, Boston, MA, USA) with EGM-2 SingleQuot growth supplements (Lonza, Madison, WI, USA). Cells that were <6 passages were used in this study. TNFα was purchased from Promega (G5421, Madison, WI, USA). The kinase inhibitors used in this study are: p38 kinase (SB202190; Sigma), CKII (TBCA; Calbiochem, San Diego, CA, USA), MSK (SB747651; Tocris Bioscience, Bristol, UK), JNKs (SP600125; Sigma); ERKs (U0126; Sigma) and MNK (CGP57380; Cayman Chemical, Ann Arbor, MI, USA; or cercosporamide; Tocris Bioscience). The siRNAs were purchased from Thermo Scientific (Waltham, MA, USA) and the siRNA target sequences are listed in Supplementary Table 1. Antibodies used in this study are listed in the Supplementary Table 3. The transfection reagents DharmaFECT1 (T-2001) and Lipofectamine 2000 (LF2000) were purchased from Thermo Scientific and Invitrogen (Grand Island, NY, USA), respectively.