Fig. 4.

IL-1β and TNF-α activate NF-κB/COX2 → HIF1-α axis in HER2-negative mammary epithelial cells. (A) HB2 cells as well as their HER2-overexpressing variants were serum-starved and treated with IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 15, 30, 60 and 120 minutes, or (B) 24, 48 and 72 h. NF-κB activation, COX2 and HIF1-α expression were analysed by Western blotting. Densitometry was done with ImageJ software. (C) HB2 and HB2(HER2+) cells were serum-starved and treated with IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 24, 48, 72 and 96 h. CAIX (downstream target of HIF1-α activity) expression was evaluated via Western blotting. (D) HB2 cells were grown with magnolol (10 µM, NF-κB inhibitor) or celecoxib (25 µM, COX2 inhibitor), ± IL-1β (10 ng/ml), ± TNF-α (10 ng/ml) and analysed for COX2 and HIF1-α expression.