Myc induces DNA damage via ROS in vivo. Comparison of freshly isolated Eμ-myc lymphoma cells and immunobead-selected nontransgenic B cells derived from an Atm^+/+ or an Atm^−/− background (n = 4 individual samples per genotype). (A) 2′-7′-Dichlorodihydrofluorescein diacetate–based flow cytometric analysis of cellular ROS levels (left); representative example visualized by fluorescence microscopy (right). Original magnification, × 200. (B) Oxidative DNA damage measured as the relative induction of mean tail moments in the COMET assay prior to and after treatment with Fpg; quantification (left); representative examples of comets (right). (C) γ-H2AX foci per cell in cytospin preparations; same cell populations stably expressing Bcl2 to block apoptosis in the right panel; quantification (left) and representative examples (right). (D) Immunoblot analysis of total Atr, Atr-P-S431, and tubulin as a loading control in nontransgenic B lymphocytes as compared with representative control and Atm^−/−Eμ-myc lymphomas. (E) Proliferation (left, cells with S-phase DNA content), ROS induction (middle, as in panel A), and γ-H2AX foci (right, as in panel C) in primary nontransgenic B cells displayed as relative values at 48 hours after LPS stimulation (50 μg/mL) compared with no LPS.