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. 2018 Sep 18;8:14012. doi: 10.1038/s41598-018-32356-8

Figure 4.

Figure 4

yH2AX spread analysis. (A) Upper panel: example of three types of cell nuclei after yH2AX immunocytochemistry regarding the number of foci (red: Alexa 594 γ-H2AX foci; blue: DAPI cell nucleus). Graph: frequency distribution of nuclei with 0 to 65 foci in increments of five (calculated from 220 nuclei per cell line; p = 0.00001 determined by two tailed t-test). Imaged with Leica SP8 X FLIM confocal microscopes (HC PL APO CS2 63 × /1.40 OIL objective). (B) Primary DNA damage in HepG2 cells estimated by the alkaline comet assay. Photomicrographs of the nuclei of HepG2 cells observed after the alkaline comet assay procedure. (a) control shCTRL cells; (b) control shCTRL cells cultivated with the addition of adenosine; (c) HepG2 cells with silenced ACHY (shAHCY), where an arrow indicates the damaged DNA that resembles comet-style features; (d) HepG2 cells with silenced ACHY (shAHCY) cultivated with the addition of adenosine. Agarose microgels were stained with ethidium bromide (20 µg/mL) and analysed under epifluorescence microscope (Olympus BX51), under 200× magnification. Photomicrographs acquired by the image analysis system Comet Assay IVTM (Perceptive Instruments Ltd., UK). (C) indicators for DNA damage were (a) tail length, (b) tail intensity, and (c) total area. For each sample, three replicates were prepared, with 100 independent comet measurements per slide, with 300 measurements performed per sample. The image analysis system ‘Comet Assay IVTM’ (Perceptive Instruments Ltd., UK), in combination with an epifluorescence microscope (Olympus BX50, Japan) equipped with appropriate filters, under 200x magnification, was used for the analysis. The results are shown as the median/mean value, and the range of the measured values (min-max); scale bar, 20 µm. Statistical significance of the data was evaluated using descriptive statistics, ANOVA with post hoc Scheffé’s test (intra-group comparisons) and the Mann-Whitney U-test (inter-group comparisons). The level of statistical significance was set at P < 0.0.05. The abbreviations above the whiskers indicate which samples differ with statistical significance. For ANOVA, the abbreviations are as follows: nc – vs. corresponding negative control; # – vs. all other samples. A sign * designates the samples that showed a statistically significant increase of the studied comet parameter compared to the related clone with regard to the addition of adenosine. shAHCY.1 – cells with silenced AHCY, replica 1; shAHCY.2 – cells with silenced AHCY, replica 2., Positive control – shCTRL cells exposed ex vivo to 50 µM hydrogen peroxide for 10 minutes on ice. For each sample, three replicate slides were prepared.