Recombinant opn protein

Some cohort cultures of A549 or U937 cultures were treated with tumour necrosis factor-alpha (TNF-a) (2 ng ml À1 ) (Sigma-Aldrich, Poole, UK). Some cohort A549 cultures were treated with OPN recombinant protein (2 mg ml À1 ; R&D Systems, Abingdon, UK) or phosphate-buffered saline (PBS) vehicle for 24 h. U937 cells were treated with a low concentration of OPN recombinant protein (0.2 mg ml À1 ; R&D Systems) or a high concentration of OPN recombinant protein (2 mg ml À1 ). Some cohort A549 cultures were treated with human OPN siRNA (SI00012201), C/EBP homologous protein (CHOP) siRNA (SI00059528), or mixed lineage kinase domain-like protein (MLKL) siRNA (SI03112249), or CD44 siRNA or scrambled siRNA (SI00012775, Qiagen, Crawley, West Sussex, UK) 6 h before experiments. The siRNA-targeting human OPN, CHOP, and MLKL were dissolved in siRNA suspension buffer and administered to A549 cells at a dose of 20 nmol L À1 ; scrambled siRNA served as a negative control. Cells were incubated with siRNA for 6 h in humidified air containing carbon dioxide 5%, after which it was removed and replaced with an experimental medium followed by TNF-a treatment.