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. 2020 Oct 16;21(20):7679. doi: 10.3390/ijms21207679

Figure 2.

Figure 2

NOX1 is required for ROS generation and neurite outgrowth induced by Y-27632. (A) PC12 cells were treated with 25 μM Y-27632 for 2 h in the presence or absence of 10 μM DPI. Microphotographs of the cells were taken, and representative images are shown in the left panel. Cells with neurite processes longer than the cell body diameter were counted, and the percentages of neurite-containing cells are shown in the right panel. Scale bar = 50 µm. Culture images are at a magnification of 20×. (B) PC12 cells were incubated with 2 μM DCF-DA and treated with 10 μM DPI. Cells were then exposed to 25 μM Y-27632 for 1 h. The fluorescence intensity was measured using a confocal microscope. Note that the NOX-1 inhibitor DPI prevented both ROS generation and neurite elongation caused by Y-27632. Scale bar = 20 µm. Culture images are at a magnification of 40×. (C) Western blot analysis of the lysates of cells transfected with empty vector (control shRNA) or shRNA targeting NOX1 (NOX1 shRNA). Gels were transferred and blotted with specific antibodies against NOX1 or α-tubulin. (D) PC12 cells transfected with control shRNA or NOX1 shRNA were treated with 25 μM Y-27632 for 2 h. Cells with neurite processes longer than the cell body diameter were counted, and the percentages of neurite-containing cells were determined. Note that knockdown of NOX1 by shRNA transfection inhibited neurite outgrowth induced by Y-27632. Scale bar = 50 µm. Culture images are at a magnification of 20×. (E) Western blot analysis of the lysates of NOX1 knockdown cells transfected with the Flag-NOX1 plasmid for 24 h. Gels were transferred and blotted with specific antibodies against NOX1, Flag or GAPDH. Note that the expression of NOX1 increased after Flag-NOX1 transfection in NOX1-knockdown cells. (F) Representative immunocytochemistry analysis of NOX1 (green) and phalloidin (red) in Flag-NOX1 transfected NOX1-knockdown cells treated with 25 μM Y-27632. Cells with neurite processes longer than the cell body diameter were counted, and the percentages of neurite-containing cells are shown in the right panel. Note that Y-27632 promoted neurite outgrowth in NOX1-knockdown cells transfected with the Flag-Nox1 plasmid. Arrowheads indicate a neurite promoted by Y-27632 from a cell transfected with Flag-Nox1. Empty arrowheads indicate a nontransfected cell that does not bear neurites. Scale bar = 20 µm. Culture images are at a magnification of 63×. The results are presented as the means ± S.E.M of at least three independent experiments. ** p < 0.01; n.s, not significant.