Figure 4. MMV652103 induces the production of reactive oxygen species (ROS) and induces double-strand DNA breaks. Levels of cellular (A) H₂O₂ and (B) GSH in MCF7 cells after 24 h treatment with ½ IC₅₀ and IC₅₀ concentrations of MMV652103. Doxorubicin at its IC₅₀ concentration (9.84 nM) for MCF7 cells was used as a control. Graphs show the mean H₂O₂ and GSH levels of three independent experiments ± SEM. (C) MCF7 and T47D cells were treated with vehicle (DMSO), ½ IC₅₀ and IC₅₀ for 24 and 48 h and western blotting performed to determine the levels of γH2AX. (D) Immunocytochemistry of MCF7 and T47D cells treated with vehicle (DMSO) and IC₅₀ for 24h. Images were captured under a confocal microscope (Zeiss, Germany) at 600X magnification and are representative of five randomly selected fields of view for each treatment (scale bars indicate 10 μM). The results were analyzed and show the average intensity (arbitrary units) for each treatment. (E) Western blot analyses of protein harvested from cells treated as indicated and incubated with antibodies against p-p38, p53 and p21. For all western blots total p38 was used as a loading control. Densitometry readings were obtained using ImageJ and protein expression levels are represented as a ratio of protein of interest/p38 normalized to the vehicle control sample. Blots are representative of at least three independent experiments. Data were analyzed using GraphPad Prism 6.0 and a parametric unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001.