Extracellular DNA Containing (dG)n Motifs Penetrates into MCF7 Breast Cancer Cells, Induces the Adaptive Response, and Can Be Expressed

Figure 3

8-oxodG levels in MCF7 and GC-DNAs. (a) 8-oxodG levels in pEGFP and pEGFP-Gn. The method of immunoassay using 8-oxodG antibodies conjugated with alkaline phosphatase was used. (1, 2) Standard samples of the oxidized genomic DNA (10 ng/dot) with a known content of 8-oxodG were applied in order to plot a calibration curve for the dependence of the signal intensity on the number of 8-oxodG copies. (3, 4) The samples of the oxidized and nonoxidized (control) pEGFP and pEGFP-Gn (10 ng/dot) were applied. MCF7: GC-DNAs after 3 hours of incubation with MCF7; UV/H₂O₂: plasmids were oxidized in 0.1% H₂O₂ solution with UV irradiation (). (b) FCA. (1) Cell plots: FL2 (8-oxodG-PE) versus FCS. (8-oxodG)⁺: gated area. Graph: relative proportions of 8-oxodG positive cells (change with time). (c) FM. Evaluation of 8-oxodG (PE, red) in the cells treated with GC-DNAs (3 h). Yellow arrows indicate the surface of the cells, where it is possible to localize the granules of oxidized DNA. (d) FM. 8-oxodG (FITC, green) and mitochondria (mitotracker TRMR, red) in the cells treated with pEGFP-Gn (1.5 h). The mitochondria were analyzed in nonfixed cells with TRMR. The cells were then fixed and analyzed for 8-oxodG. Magnification ×200.

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