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. 2016 Jun 14;291(33):17303–17318. doi: 10.1074/jbc.M116.721936

FIGURE 6.

FIGURE 6.

The NTBI-derived iron export activity of FPN1 was suppressed by PCBP2 silencing in mammalian cells. A, PCBP2 silencing decreases FPN1-dependent iron export. HEp-2 cells stably expressing DMT1A-I were cultured in serum-free DMEM and preloaded with 100 μm FAC or 100 μm Fe-NTA for 6 h at 37 °C. The cells were washed with PBS, transfected with FPN1-GFP expression plasmid or GFP and PCBP2 siRNA or control siRNA, and then incubated in serum-free RPMI 1640 medium for 18 h at 37 °C. The cells were then lysed in TNE buffer for immunoblotting analysis. The siRNA are denoted as: −, non-siRNA; N, negative control siRNA; P2, PCBP2 siRNA. Similar results were obtained from at least three independent experiments. B, 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 a quantitative datum of band imaging appearing in A in order from left to right. Each column is indicated as follows: 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) and GFP(P2): post-transfection 18 h siRNA(PCBP2), plasmid (GFP). Significance was determined by Student's t test: †, p < 0.05; §, p < 0.001. C, the histograms show the relative intracellular iron level. The cells were treated as described in A 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 using Student's t test: †, p < 0.05; §, p < 0.001.

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