FIGURE 7.
The TBI-derived iron export activity of FPN1 was suppressed by PCBP2 silencing in mammalian cells. A and C, FPN1-GFP exports iron loaded as TBI. HEp-2 cells were cultured in serum-free DMEM and preloaded with 0.3 mg/ml holo-Tf for 12 h at 37 °C. The cells were washed with PBS, transfected with FPN1-GFP expression plasmid, and then incubated in serum-free RPMI 1640 medium for 18 h at 37 °C. The cells were lysed in TNE buffer for immunoblotting analysis (A) or in 50 mm NaOH (C) for a ferrozine-based iron assay. B, the density of specific bands from immunoblotting was measured with a computer-assisted imaging analysis system (NIH ImageJ), and normalization was to total protein loaded. The histogram shows the expression ratio of PCBP2, TfR1, tubulin, FPN1, GFP, and ferritin. Each column corresponds to an immunoblotting analysis lane in A in order from left to right. Each column is denoted in the following way: (−)12 h: holo-Tf(-)-incubation 12 h; (+)12 h: holo-Tf(+)-incubation 12 h; (+)18 h: holo-Tf(+)-incubation 12 and 18 h; (+)18 h FPN/GFP: holo-Tf(+) incubation 12 h; and 18 h FPN1 or GFP transfection. Significance was determined using Student's t test: †, p < 0.05; §, p < 0.001. C, the histogram shows the relative intracellular iron level. The detailed method is described under “Experimental Procedures.” The results in the histogram are presented as the means ± S.E. of experiments performed in triplicate. Significance was determined by Student's t test: ‡, p < 0.01; §, p < 0.001. D, PCBP2 silencing decreases FPN1-mediated iron export. HEp-2 cells were cultured in serum-free DMEM and preloaded with 0.3 mg/ml holo-Tf for 12 h at 37 °C. The cells were washed with PBS, transfected with FPN1-GFP expression plasmid and PCBP2 siRNA, and then incubated in serum-free RPMI 1640 medium for 18 h at 37 °C. The cells were lysed in TNE buffer for immunoblotting analysis. The symbols used to describe the siRNAs are as follows: −, non-siRNA; N, negative control siRNA; and P2, PCBP2 siRNA. Similar results were obtained from at least three independent experiments. E, the density of specific bands from immunoblotting was measured with a computer-assisted imaging analysis system (NIH ImageJ). The histogram shows the expression ratio of PCBP2, TfR1, tubulin, FPN1, GFP, and ferritin. Each column corresponds to an immunoblotting analysis lane in D in order from left to right. Each column is denoted in the following way in each band imaging of PCBP2, TfR1, Ferritin, Tubulin, FPN1 and GFP: 0 h: post-transfection 0 h siRNA (−), plasmid (−); FPN(−): post-transfection 18 h siRNA (−), plasmid (FPN1); FPN(N): post-transfection 18 h siRNA (negative control), plasmid (FPN1); FPN(P2): post-transfection 18 h siRNA(PCBP2), plasmid (FPN1); GFP(−): post-transfection 18 h siRNA(−), plasmid (GFP); GFP(N): post-transfection 18 h siRNA (negative control), plasmid (GFP); GFP(P2): post-transfection 18 h siRNA(PCBP2) and plasmid (GFP). Significance was determined using Student's t test: †, p < 0.05; §, p < 0.001. F, the histogram shows the relative intracellular iron level. Cells were treated as described in D and lysed in 50 mm NaOH. The iron concentration was measured using a ferrozine-based iron assay. The detailed method is described under “Experimental Procedures.” The results in the histogram are presented as the means ± S.E. of three independent experiments. Significance was determined by Student's t test: ‡, p < 0.01; §, p < 0.001.