Phorbol 12 myristate 13 acetate pma

Cells were seeded in full growth medium (10% FBS, 1X MEGS DMEM:F12) for 24 h prior to replacing the medium. Briefly, the cells were washed and the medium was replenished using the components in 10% FBS DMEM:F12 as indicated in figure legends. Cells were treated using 3 ng/ml EGF (Sigma), 0.01 µg/ml IGF1 (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 0.4% v/v BPE (Thermo Fisher Scientific) and TGFβ-1 recombinant protein (Thermo Fisher Scientific). The TGFβR inhibitor SB431542 (SelleckChem) was used at 5 µg/ml, 25 µM of PI3K inhibitor LY294002 (SelleckChem), 10 µM of AKT inhibitor MK-2206 (SelleckChem), 100 nM of FGFR inhibitor BGJ398 (SelleckChem) and 1 µg/ml PKC activator Phorbol 12-myristate 13-acetate (PMA; SelleckChem). The bFGF neutralizing antibody, clone bFM-1 (Millipore), was used at 2.5 µg/ml.

A549, H1437, H1373, H838, H23, H322, HCC827, H1650, and H1975 cells were obtained from American Type Culture Collection, PC9 cells from European Collection of Authenticated Cell Cultures, and II-18 cells from RIKEN BRC cell bank. Human monocytic THP-1 cells were kindly provided by Y. Mori and K. Akashi (Kyushu University). These cell lines were cultured in RPMI 1640 medium (Gibco) or Dulbecco’s modified Eagle’s medium (Gibco), each supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Phorbol 12-myristate 13-acetate (PMA) (#S7791) was obtained from Selleckchem, and dissolved in dimethyl sulfoxide. Recombinant human IL-2 (R&D Systems, #202-IL) was dissolved in sterile deionized water. Recombinant human IL-1β (R&D Systems, #201-LB) and recombinant human IFN-γ (Peprotech, #300-02) were dissolved in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. Fludarabine (#S1491), BAY 11-7082 (#S2913), SP600125 (#S1460), and U0126 (#S1102) were obtained from Selleckchem, and SB203580 was from Invivogen (#tlrl-sb20). Each inhibitor was dissolved in dimethyl sulfoxide. The concentration of each inhibitor was determined based on product data sheets and previous reports (8 (link), 9 (link)).