Fig. 7
Pre-treatment with ROS scavenger completely blocked the cytotoxic and genotoxic effect of ZOT5-1-Me and ZOT5-1-Et in U-87 MG cells. (A) Pre-incubation with N-acetyl-l-cysteine (NAC, 1 mM, 1 h) decreased intracellular reactive oxygen species (ROS) induced by ZOT5-1-Me and ZOT5-1-Et in U-87 MG as estimated by CM-H2DCFDA assay. The data were normalized to control cells (CTRL, 100%, n = 6). (B) Pre-incubation with NAC restored cell viability of U-87 MG cells after 48 h treatment with ZOT5-1-Me and ZOT5-1-Et as evaluated by CCK-8 assay. The data were normalized to control cells (CTRL, 100%, n = 6). (C) Pre-incubation with NAC reduced the externalisation of phosphatidylserine in U-87 MG cells as evaluated by Annexin V/PI assay followed by flow cytometry (FACS) quantification (n = 6). (D) Representative FACS dot plots after 48 h treatment with ZOTs are presented with the indicated percentages of necrotic (Q1), late-apoptotic (Q2), early-apoptotic (Q3), and viable cells (Q4). Apoptosis was presented as a percentage of Annexin V-positive cells (Q2 + Q3). (E) Pre-incubation with NAC diminished ZOT5-1-Me and ZOT5-1-Et-evoked apoptosis in U-87 MG cells as determined by Caspase-Glo 3/7 assay (n = 6). The data were normalized to control cells (CTRL, 100%, n = 6). (F) Pre-incubation with NAC decreased DNA damage (percentage of DNA in the comet tail) induced by ZOT5-1-Me and ZOT5-1-Et in U-87 MG cells as determined by the alkaline versions of comet assay (n = 100). (G) Representative images of comets from the alkaline version of the comet assay are presented. Results are presented as bar plots with mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ns – not statistically significant.
