Figure 5.

LCN-2 knockdown in primary human MΦ attenuates the iron-release phenotype upon co-culture with breast cancer cells. Primary human MΦ were transfected with siRNA targeting LCN-2 (siLCN-2) or a scrambled control siRNA (sictrl). 24 h post-transfection, cells were co-cultured with MCF-7 cells for 48 h. (A) mRNA expression of iron-regulated genes (FPN, HAMP, FTL) in sictrl (left panel) or LCN-2-depleted (right panel) MΦ after co-culture (solid bars) was analyzed by qPCR and normalized to 18 S expression (n = 10–19). Changes were determined relative to the respective expression in naïve control MΦ (ctrl, open bar). (B) Protein expression of FPN (surface, left panel) and FTL (intracellular, right panel) in sictrl (open bars) or LCN-2-depleted (solid bars) MΦ after co-culture was determined by flow cytometry, normalized to the expression in naïve, sictrl transfected MΦ (n = 11–21). (C) Iron amount in the supernatant of sictrl (open bars) or LCN-2-depleted (solid bars) MΦ after co-culture was quantified by AAS and normalized to supernatants of naïve, sictrl transfected MΦ (n = 4). (D) Proliferation of MCF-7 cells upon stimulation with supernatant (MΦ-SN) from LCN-2-depleted MΦ co-culture (co-cu) was determined on an xCELLigence instrument and is given relative to MCF-7 cells treated with supernatant from naïve, sictrl transfected MΦ (n = 4). Data are means ± SEM from independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the respective controls unless indicated otherwise.