Fig. 7.

Macrophages-conditioned medium (M1-CM) promotes proliferation of HER2-negative mammary epithelial cells via activation of the NF-κB/COX2 → HIF1-α axis. (A) THP-1 cells were differentiated into M1- and M2-like macrophages as described in material and methods. Verification of THP-1 differentiation, i.e., expression of specific markers CD68 and iNOS for M1-like macrophages, CD163 for M2-like macrophages, was done by fluorescent microscopy. (B) HB2 and their HER2-overexpressing counterparts were cultured in 3D matrigel for 8 days in the presence of M1-like-conditioned medium (CM-M1). (C) HB2 cells were serum-starved and treated with CM-M1. NF-κB activation, COX2 and HIF1-α expression were analysed by Western blotting. (D) HB2 cells were grown in 3D matrigel for 8 days and treated with M1-like-conditioned medium ± magnolol (10 µM) or celecoxib (10 µM). Representative images were taken, an average colony size was determined with ImageJ software. Data are expressed as means ± SD (n = 3), ***p < 0.001.