NOX5 inhibition impairs MAPK and NFκB pathways during Mo-DC differentiation. Representative western blot analysis (n = 3) of (A) Mos treated with NAC (10 mM), DPI (0.1 µM), CEL (2 µM), or EBS (40 µM) for 15 minutes prior to addition of cytokine differentiation stimuli (GM-CSF/IL-4) for 6 days. On day 7, cells were harvested and lysed. (B) Mos were treated for 18 hours with GM-CSF/IL-4 to induce NOX5 expression and then transfected with 200 nM scrambled or NOX5 siRNA. Blots were incubated with (left) anti-phospho ERK, anti-phospho p38, anti-phospho p65, anti-phospho STAT5, or (right) with antibodies against the total proteins. Blot quantification can be found in supplemental Figure 7A-B. (C) Our model of regulation of Mo-DC differentiation shows that the complex located on the outer membrane of the mitochondria releases ROS that positively regulate, directly or indirectly, the cytokine-induced JAK2 phosphorylation, leading to subsequent activation of STAT5 phosphorylation, which then dimerize and translocate to the nucleus, promoting cell differentiation. In parallel, JAK2 activation will also induce MAPKinase and AKT/NFκB activation. These signaling events promote differentiation into DCs. Scr, scrambled.