FIGURE 10.
Silencing of MnSOD expression enhances Ang II-induced decrease in NO bioavailability and fibronectin expression in MCs. A, MCs were transfected with Scr or siMnSOD MCs, and expression of MnSOD was assessed in total cell lysate (Tot) by Western blot analysis. Immunoblotting with MnSOD antibody confirmed the silencing of the enzyme by siMnSOD. B, after transfection with Scr or siMnSOD, mitochondrial ROS production in response to Ang II (1 μm; 10 min or 1 h) was evaluated using MitoSOX Red fluorescence and confocal microscopy. Fluorescence intensity was semiquantified, and the values are the means ± S.E. from three independent experiments. **, p < 0.01 versus control cell transfected with Scr. ##, p < 0.01 versus Ang II in cells transfected with Scr. §§, p < 0.01 versus control cell transfected with siMnSOD. C, MCs were transfected with Scr or siMnSOD and treated with Ang II (1 or 24 h), and cGMP synthesis was determined. The values are the means ± S.E. from three independent experiments. **, p < 0.01 versus Scr-transfected cells. #, p < 0.05 versus Ang II in cells transfected with Scr. §§, p < 0.01 versus control cell transfected with siMnSOD. D and E, after transfection with Scr or siMnSOD, fibronectin protein expression in response to Ang II (1 μm; 24 h) were evaluated by Western blot analysis (D) and immunofluorescence (E). D, right panel, histogram representing the ratio of the intensity of fibronectin bands quantified by densitometry factored by the densitometric measurement of GAPDH band. The data are expressed as percentages of control (cells transfected with Scr), where the ratio in the control was defined as 100%. The values are the means ± S.E. from three independent experiments. E, right panel, histogram representing the semiquantification of fluorescence intensity. The photomicrographs are representative of three individual experiments. **, p < 0.01 versus control cells transfected with Scr. ##, p < 0.01 versus Ang II in cells transfected with Scr. §§, p < 0.01 versus control cell transfected with siMnSOD.