Human lung carcinoma type 2 epithelium-like A549 cells, purchased from Riken BioResource Center Cell Bank (Tsukuba, Ibaraki, Japan), were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Life Sciences, MO, USA) with 10% heat-inactivated fetal bovine serum (FBS; GE Healthcare Japanhealth, Tokyo, Japan), 100 U/ml penicillin, 100 mg/ml streptomycin in 10 cm dishes at 37 °C in a humidified atmosphere of 5% CO2. A549 cell line has been characterized as an in vitro model of alveolar type 2 pneumocytes of the human lung because of capacity of secreting lung surfactant-associated glycoproteins [8 (link)].
Reagents were purchased commercially as follows: human IL-1β (Humanzyme, Chicago, IL, USA); human TNF-α (Prospec, Rehovot, Israel), human IFN-γ (Peprotech, NJ, USA); dexamethasone, Fas ligand (super Fas Ligand) (Enzo, PA, USA); RIPA buffer (Thermo Fischer Scientific, Tokyo, Japan); lipopolysaccharide (LPS: E. coli O111:B4, Sigma-Aldrich Life Sciences); rapamycin, parthenolide, SP600125, SB203580, U0123, LY294002, and QVD-OPh (Cayman Chemical, MI, USA).
Antibodies were as follows: anti-iNOS (R&D systems, MN, USA); anti-HO-1 (Enzo); anti-beta actin (MBL, Nagoya, Japan); anti-JNK (Abcam Japan, Tokyo, Japan); anti-cox-2 (BD Japan, Tokyo, Japan); anti-ICAM-1 (Santa Cruz Biotechnology, TX, USA). All of the other antibodies were purchased from CST Japan, Tokyo, Japan.
Reagents were purchased commercially as follows: human IL-1β (Humanzyme, Chicago, IL, USA); human TNF-α (Prospec, Rehovot, Israel), human IFN-γ (Peprotech, NJ, USA); dexamethasone, Fas ligand (super Fas Ligand) (Enzo, PA, USA); RIPA buffer (Thermo Fischer Scientific, Tokyo, Japan); lipopolysaccharide (LPS: E. coli O111:B4, Sigma-Aldrich Life Sciences); rapamycin, parthenolide, SP600125, SB203580, U0123, LY294002, and QVD-OPh (Cayman Chemical, MI, USA).
Antibodies were as follows: anti-iNOS (R&D systems, MN, USA); anti-HO-1 (Enzo); anti-beta actin (MBL, Nagoya, Japan); anti-JNK (Abcam Japan, Tokyo, Japan); anti-cox-2 (BD Japan, Tokyo, Japan); anti-ICAM-1 (Santa Cruz Biotechnology, TX, USA). All of the other antibodies were purchased from CST Japan, Tokyo, Japan.