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. 2016 Jan 20;291(11):5688–5707. doi: 10.1074/jbc.M115.669952

FIGURE 9.

FIGURE 9.

A and B, effect of MIOX overexpression on TGF-β1 expression and bioactivity under HG ambience in LLC-PK1 cells. No significant increase in the expression or bioactivity was observed with transfection of EV (pcDNA) under basal LG conditions (columns 1 and 2). HG ambience induced a moderate increase in the bioactivity and expression of TGF-β1, and no significant further increase was seen with EV transfection (columns 3 and 4). Interestingly, transfection of MIOX-pcDNA under LG ambience increased the bioactivity and expression of TGF-β1 comparable with those seen under HG ambience (column 5). Overexpression of MIOX in the presence of HG markedly increased the bioactivity and expression of TGF-β1, and it was significantly reduced with MIOX siRNA treatment (columns 6 and 7). C–E, modulation of fibronectin expression by MIOX under high glucose ambience. Under LG ambience, fibronectin expression was quite low in the “cell culture medium” with no significant change following EV transfection (C, columns 1 and 2). Under HG ambience, a ∼3-fold increase in fibronectin expression was seen, which was unaffected by EV transfection (C, columns 3 and 4). MIOX-pcDNA transfection in LG ambience also caused a comparable increase in fibronectin expression (C, column 5), whereas MIOX-pcDNA transfection under HG ambience caused a marked increase in fibronectin expression, which was significantly reduced by MIOX siRNA treatment (C, columns 6 and 7). At the cellular level, a low level of fibronectin expression was observed under LG ambience, and no significant change was seen in cells transfected with EV (D (a)). A moderate increase in expression of fibronectin was observed in cells under HG ambience (D (b)). A marked increase in fibronectin expression was observed with MIOX overexpression under HG ambience (D (c)), which was restored to basal levels following MIOX siRNA treatment (D (d)). In order to affirm that MIOX-mediated fibronectin expression was modulated via ROS, the cells were treated with NAC. With the treatment with NAC, expression of fibronectin was reduced in cells transfected with MIOX as well as in those concomitantly exposed to HG ambience, confirming that MIOX transfection alone increased the fibronectin expression via ROS (D, a and c versus b and d) (n = 4; *, p < 0.01; #, p < 0.05).