Genome instability in MCF-7 cells exposed to gDNA^OX at final concentration 50 ng/mL for 24 hours.

<div><p>A – multiple micronuclei [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>], chromatin bridges [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>], M-phase chromatin decondensation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>], non-treated control cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B4" target="_blank">4</a>] (x100). </p> <p>B – proportions of cells with micronuclei in non-treated control cells, cells exposed to gDNA, cells exposed to gDNA<b>^OX</b>. Grey columns: non-confluent, actively proliferating MCF-7 culture. Black columns: MCF-7 cells at high confluency. *p < 0.05 against control group of cells, non-parametric U-test.</p> <p>С - Exposure to gDNA<b>^OX</b> (50 ng/mL, 2 hours) induces formation of 8-oxodG-containing micronuclei (x100). </p></div

The exposure to gDNA^OX (50 ng/mL) leads to a transient increase in expression cytoplasmic DNA sensor AIM2, while not changing expression levels of TLR9.

<div><p>A - intracellular localization of AIM2 (FITC-conjugated antibodies) and labeled probe gDNA<b>^red-ox</b> (x40). B – the ratio of the levels of AIM1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and TLR9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] – encoding RNAs to the levels TBP-encoding reference mRNA in cells exposed to gDNA or gDNA^OX for 2 hrs (grey columns) and 48 hrs (black columns).</p> <p>C and D – Flow cytometry detection of AIM2 (C) and TLR9 (D) expression in MCF-7. Cells were stained with AIM2 (C) or TLR9 (D) antibody (secondary PE-conjugated antibodies). Panels D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and E [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] – control cells plots: FL2 versus SSC. R: gated area. Panels C [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] and D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]: median signal intensity of FL2 (R) in MCF-7 cells (mean value for three independent experiments). Panels C [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>] and D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]: relative proportions of AIM2- or TLR9-positive cells in R gates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. Background fluorescence was quantified using PE-conjugated secondary antibodies. </p> <p>*p < 0.05 against control group of cells, non-parametric U-test.</p></div

The exposure to gDNA^OX leads to an increase in the production of ROS.

<p>А – Microscopy-based evaluation of MCF-7 cells sequentially treated with DNA (50 ng/mL) and H2DCFH-DA (control, gDNA, gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]) and incubated for 30 minutes (x100). Alternatively, MCF-7 cells were incubated with DNA (50 ng/mL) for 1 hour followed by addition of H2DCFH-DA and photography 30 minutes later (gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]). B - MCF-7 cells exposed to gDNA^ox (0.5h; 50ng/mL), were sequentially treated with Mito-tracker TMRM (15 min) and H2DCFH-DA (15 min) (x200). C - Co-detection of labeled probe gDNA^red (50 ng/mL) and DCF after 30 minutes of incubation. D - The results of the quantification of fluorescence using plate reader [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. The time kinetics of fluorescence outputs in cells sequentially treated with H2DCFH-DA and, three minutes later, a DNA sample at final concentration of 5 or 50 ng/mL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. The same for cells pretreated with DNA (final concentration 5 ng/mL) for one hour, with subsequent addition of H2DCFH-DA. *) p < 0.05 against control group of cells, non-parametric U-test.</p

Cell death in MCF-7 cultures exposed to either gDNA or gDNA^OX at final concentration 50 ng/mL for 48 hours.

<div><p>A. Total number of cells in studied cell population.</p> <p>B. (FACS) – enumeration of cells with sings of early apoptosis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. - the distribution of fluorescence intensities of the cells stained with Annexin V-FITC (green color) или FITC-conjugated secondary antibodies (grey color) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - control cells plots: FL1 versus SSC. R: gated area [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]. - the proportion of Annexin V -positive cells in total cell population. </p> <p>C. Evaluation of modified nuclei in three studies typed of MCF-7 cultures. (1) -Example of Hoechst33342 staining; (2) - Graph of the proportion of cells with modified nuclei in three studied types of MCF-7 cultures. </p> <p>D. Electrophoresis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and evaluation of ecDNA concentrations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] in the media of non-treated control cells and cells exposed to either gDNA or gDNA^OX. Dashed line indicates amounts of ecDNA that should be present in the media when exogenous DNA is taken into account. *p < 0.05 against control group of cells, non-parametric U-test.</p></div

The analysis of 8-oxodG content in cells exposed to either gDNA or gDNA^OX (50 ng/mL).

<p>A - Cells stained with PE-labeled anti-8-oxodG antibodies and DAPI (x20). B - Three types of anti-8-oxodG stain distribution observed in cells treated with gDNA<b>^OX</b> (x100). Cell were incubated with DNA samples for 1 hour, fixed with 3% formaldehyde, permeated with 0,1 % triton X100 and stained with anti-8-oxodG (PE-conjugated secondary antibodies). C – colocalization of 8-oxodG with mitochondria. Cells were incubated with <b>gDNA^OX</b> for 0.5 hour, обработаны Mito-tracker (30 nM, 15 min), photographed, then fixed with 3% formaldehyde, permeated with 0,1 % triton X100, stained with anti-8-oxodG antibodies (FITC-conjugated secondary antibodies) and photographed again. D - 8-oxodG content in DNA exposed cells pre-treated with NAC (FACS analysis). Cells were incubated with NAC (0.15 mM) for 30 min, then exposed to gDNA^OX for 1 hour and analyzed using anti-8-oxodG antibodies (PE-conjugated secondary antibodies). Background fluorescence was quantified using PE-conjugated secondary antibodies. E - Relative proportions of nuclei stained for 8-oxodG in non-treated control cells, cells exposed to gDNA, cells exposed to gDNA^OX (grey columns). Light grey column reflects cells pre-treated with NAC and exposed to gDNA^OX. *p < 0.05 against control group of cells, non-parametric U-test.</p

Increase in activity of transcriptional factor Nf-kB in MCF-7 cells exposed to gDNA^OX at final concentrations of 50 ng/mL for 2 hours.

<div><p>A Fluorescent microscopy of cells stained with anti-p65 (FITC) antibodies (x40). B Graph of the proportion of cells with nuclear staining for Nf-kB in three studied types of MCF-7 cultures.</p> <p>C, D (FACS) - the average signal intensity of FL1 (p65) in cells stained with anti-p65 (C) and Ser529-phosphorylated р65 (D) antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. - distribution of fluorescence intensities of the cells stained with Ser529-phosphorylated р65 antibodies (FITC) (green color) или FITC-conjugated secondary antibodies (grey color) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - proportion of Ser529-phosphorylated р65 -positive cells in total cell population [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]. - the average of the median signal intensities of FL1 (Ser529-phosphorylated р65 +). Cells were cultivated either in absence (dark grey columns) or in presence of 0.15 mM NAC (light grey columns). </p></div

Activity of STAT3 is stimulated in MCF-7 cells exposed to either gDNA or gDNA^OX at final concentrations of 50 ng/mL.

<div><p>A FACS: Frequency plot for fluorescence intensities in cells stained with anti-STAT3 antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and the average of the median signal intensities of FL1 (STAT3) in these cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>].</p> <p>B Fluorescent microscopy of cells stained with STAT3 antibodies (x20) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. - non-treated control cells and cells exposed to either gDNA or gDNA<b>^OX</b> for 2 hours [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - cells pre-treated for 30 min by 0.15mM NAC, then exposed to either gDNA or gDNA<b>^OX</b> for 2 hours.</p> <p>С [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] - evidence for nuclear localization of STAT3 (x100), the nuclei were stained with DAPI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - to evaluate the background, the cells were treated with normal rabbit IgG and FITC-conjugated secondary antibodies. </p></div

Decrease in activity of transcriptional factor NRF2 in MCF-7 cells exposed to gDNA^OX at final concentrations of 50 ng/mL for 2 hours.

<p>A FACS: the average of the median signal intensities in cells stained with anti-NRF2 antibodies after various exposures. B - Fluorescent microscopy of cells stained to NRF2 (x40). C - Graph of the proportion of cells with nuclear staining for NRF2 in three studied types of MCF-7 cultures. *p < 0.05 against control group of cells, non-parametric U-test.</p

DNA damage in cells exposed to either gDNA or gDNA^OX at final concentration 50 ng/mL for 30 min and 2 hours.

<div><p>А – comet assay in alkaline conditions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. - Digital photography of the nuclei with varying degree of DNA damage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]; - cumulative histograms for tail moment and percentage of DNA within tails. The reliability of differences with the control in the obtained distributions was analyzed by means of Kolmogorov–Smirnov statistics (the table shows the values of <b>D</b> and <b>α</b>).</p> <p>B - dsDNA breaks in cells exposed to gDNA<b>^OX</b> (50ng/mL, 1 hour).Cells were processed for immunofluorescence staining with anti γH2AX antibody (x40) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>].- Three detected types of nuclei are denoted by numbers: 1- nucleus with multiple dsDNA breaks, 2- nucleus with a few dsDNA breaks, 3- nucleus with intact DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - Example of a micronucleus with dsDNA breaks.</p> <p>С – FACS analysis of γ-foci A: there main fractions of the cells as evident in gating areas R1, R2, R3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>], the distribution of γH2AX fluorescence intensities [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>], relative proportions of cells within gating areas R1-R3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]. *p < 0.05 against control group of cells, non-parametric U-test.</p></div