GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

Figure 2

Exposure of haMSCs to different extracellular DNA samples induces short-term oxidative stress. (a). (A) FACS analysis showing the homogeneity of the cells (FSC-SSC diagram) and the stained cellular fractions (SSC-FL1(DCF) diagram). Control haMSC were treated with 10 μM H2DCFH-DA; (B) FACS analysis showing the distribution of haMSC stained with DCF. Cells were pretreated with DNA samples (20 ng/mL) for 5 min and then with 10 μM H2DCFH-DA for 20 min. Gate R encircles the fraction of haMSCs that express ROS. For comparison, the figure shows the data for cells treated with 0.2 mM peroxide; (C) the kinetics of changes in the average intensity of the cells in presence of various DNA samples (20 ng/mL) or 0.2 mM peroxide. (b) (A) the kinetics of changes in (fluorescence reader, = 487 nm and = 526 nm). DNAs (20 ng/mL) or 0.2 mM H₂O₂ were added after 3 min incubation of MSC with 10 μM H2DCFH-DA. The measurements were carried out 5, 10, 15, and 20 min after addition. Data points were averaged and represented as mean ± SD for eight biological replicates. Background fluorescence values ​​of culture medium in the presence of DNAs or 0.2 mM H₂O₂ were taken into account; (B) dependence on the concentration of the added DNAs (incubation time: 20 min). .

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