Mutant Ras promotes ROS production via activation of NOX family oxidases. (A) Superoxide production by N-Ras^G12D–transduced murine Sca-1⁺ cells from either wild-type (Nox2^+/+) or Nox2-deficient (Nox2^−/−) mice was measured using Diogenes. Bar chart represents total superoxide produced during assay. (B) Mitochondrial superoxide was measured using MitoSOX. Figure depicts percentage of control or H-Ras^G12V cells positive for MitoSOX fluorescence after labeling. A PBS control was used to define positive threshold. (C) Representative histograms depict MitoSOX fluorescence in control and H-Ras^G12V cells labeled with MitoSOX (i) alone or (ii) in the presence of 5μM FeTCPP or 50μM rotenone. (D) Using Diogenes, superoxide production by CD34⁺ H-Ras^G12V cells was measured in the presence of 50μM DPI, 50μM apocynin, or 5μM rotenone (n = 3). Bar chart represents total superoxide produced. (E) Transduced CD34⁺ cells were incubated with anti–NOX2-PE and analyzed by flow cytometry. Typical histogram showing anti–NOX2-PE expression; bar chart (inset) indicates percentage of fluorescent cells after incubation with anti–NOX2-PE. Isotype control antibody was used to define positive threshold (n = 5). (F) Cytosol/membrane fractionated lysates were analyzed by Western blot using antibodies recognizing p67^phox, p40^phox, or Rac. Tubulin acted as a loading control and cytosolic marker. CD45 was used a membrane marker. C indicates cytosol; and M, membrane. Data represent mean + 1 SD. Statistical significance was calculated by ANOVA using Tukey honestly significant differences or Mann-Whitney test. †P < .001; *P < .05.