Overexpressing Nox1 and Nox4 increases ROS production.
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Total RNA was collected from F9 cells transfected with an empty vector (EV) control, pcDNA3.1-Nox1 or pcDNA3.1-Nox4 and cultured 4 days. (A) Nox1 and (B) Nox4 expression following transfection of pcDNA3.1-Nox1 or pcDNA3.1-Nox4, respectively, and relative to EV transfected cells. (C) NOX1 and (D) NOX4 protein levels following transfection of pcDNA3.1-Nox1 or pcDNA3.1-Nox4, respectively, relative to EV transfected cells. (E) ROS production using Amplex Red in F9 cells transfected with EV and treated with DMSO or RA, or transfected with pcDNA3.1-Nox1 or pcDNA3.1-Nox4. A total of 3 independent experiments were analyzed and results are presented as mean ± SEM. * denotes significance (P < 0.05) tested by a Student’s t-Test, whereas letters denote groups of significance (P < 0.05) tested by a One-Way ANOVA followed by a Tukey’s test.