Superoxide production in primary AML blasts is driven by NOX family oxidases. (A) Superoxide produced by primary AML blasts (n ≥ 10) was measured using Diogenes in the presence of NOX family inhibitors (DPI, 2-AP) or modulators of mitochondrial ROS (antimycin A, rotenone, MitoTempol). Data were normalized to superoxide output of cells alone (without additions). (B) Expression of NOX2 in normal CD34⁺ cells or primary AML blasts (background subtracted mean fluorescence) as a function of superoxide production (total photons) detected by Diogenes. The shaded gray region indicates AML samples in the lowest quartile for NOX2 expression and overproducing ROS (> mean + 2 SD of control cell superoxide production). (C) Expression of NOX1, NOX2, and NOX4 in NOX2-low, superoxide-high samples (shaded region in B) was compared with that of samples with high NOX2 expression. NOX2 values represent the background subtracted mean fluorescence (scale = ×10³); superoxide values represent log₁₀ of the total superoxide production measured by Diogenes. GAPDH expression indicates relative protein loading. The significance of difference was determined by the Mann-Whitney U test; the significance of correlation was analyzed using Spearman’s rank correlation test. *P < .05, **P < .01, ***P < .001.