Knockdown of GMFG in macrophages increases intracellular iron content. (A) Representative images of immunofluorescence analysis of TfR1 in RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours. TfR1 was visualized using anti-TfR1 antibody and Alexa Fluor 488–conjugated secondary antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Scale bar, 50 μm. Quantification (right) of immunofluorescence intensity (left). Data represent the normalized mean corrected fluorescence intensity (MFI) ± standard deviation of 3 independent experiments. (B) Flow cytometry analysis of cell surface expression levels of TfR1 (CD71) in RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 48 hours. Representative histogram of unstained control siRNA-transfected cells (gray) or cells stained with the anti-CD71 antibody in control siRNA-transfected cells (red) and GMFG siRNA-transfected cells (blue) in control siRNA (left). Quantification (right) of CD71 cell surface expression flow cytometry results (left). Data represent the normalized MFI ± SD of 3 independent experiments. (C) Representative images of immunofluorescence analysis of Alexa Fluor 568–conjugated Tf internalization in RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 48 hours. Scale bar, 50 μm. (D) Analysis of total intracellular iron levels in RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 72 hours as measured by inductively coupled plasma mass spectrometry. Iron content was normalized to the total iron in control siRNA-transfected cells. (E) Analysis of labile iron pool in RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 72 hours as measured by calcein AM. Iron content was normalized to the total iron in control siRNA-transfected cells. Data represent the mean ± standard deviation of at least 3 independent experiments. *P < .05 compared with control siRNA-transfected cells.