Figure 2
DNA-binding and ROS-inducing properties of D112. (a) (Top) Excitation and emission spectra of D112. (Bottom) Emission fluorescence intensity of D112 in the presence of DNA. (b) D112-induced ROS production in yeast as indicated by CellROX Green fluorescence. Scale bar, 100 μm. (c) D112-induced ROS production in mammalian cell lines assessed by flow cytometry. Normalized ROS level was MFI of (treated cells/untreated cells). (d) Measurement of oxygen consumption in the presence of D112 at indicated concentrations. Routine: the cellular respiration in the presence of endogenous substrates. LEAK: resting respiration after Oligomycin addition to inhibit ATP synthase. ETS: maximal electron transport capacity determined by the stepwise addition of the uncoupler FCCP. (e) Representative trace of oxygen consumption (flux) in the absence or presence of D112 pretreatment of permeabilized cells. Arrows indicate time of titration of the substrates and inhibitors. Ama, antimycin a; Asc, ascorbate; Azd, sodium azide; CI, Complex I respiration; CI+II, Complex I and II respiration; CII, Complex II respiration; CIV, Complex IV respiration; Cyt, cytochrome c; Dig, digitone; M, malate; OXPHOS, oxidative phosphorylation; P, pyruvate; S, succinate; Rot, rotenone; TMPD, tetramethyl-phenylenediamine. (f) Quantification of oxygen flux from e. All values represent the mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001
