Fig. 8: Effect of iron on IFN-γR2 internalization and macrophage polarization.

A Double fluorescence staining for IFN-γR2 and Rab5a. Totally four experimental groups were prepared: cells were cultured in serum-free culture medium as the control group 1, in serum-containing complete culture medium as the control group 2. In parallel, to evaluate iron concentration on the IFN-γR2 distribution, complete medium supplemented with 10 μM deferoxamine (DFO) to remove free ferric iron (Fe³⁺) and decrease serum ferritin levels as group 3. Serum-free medium supplemented with 10 μM ammonium ferric citrate (FAC) to increase the iron concentration as group 4. THP-1-derived macrophages were cultured for 24 h in this medium and subsequently stimulated with LPS + IFN-γ for 3 h. Then, double fluorescence staining for IFN-γR2 and Rab5a was performed. A1. Representative fluorescence images. Scale bars, 50 μm. A2. The colocalization of internalized IFN-γR2 with Rab5a was calculated (n = 5). B Western blot analysis of the effect of iron on the activation of STAT1 during macrophage polarization. B1. Representative western blot analysis. B2. Quantitation of protein levels in (B1) (n = 3).