Fig 1. Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs).

(A) Results of the immunocytochemical analysis and the method of tissue printing. (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V. faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).