Fig. 3.

DNA damage and apoptosis in the presence of hydroxyurea and MS-275. a Flow cytometry analysis of HCT116 cells using Annexin-V-FITC staining. Cells were treated with 1 mM hydroxyurea (HU), 2 µM MS-275 or both for 24–48 h. Results represent the mean ± SD (n = 3; one-way ANOVA, **P < 0.01). b HCT116 cells were treated with 1 mM HU and 2 µM MS-275 for 6 and 24 h. PARP1 cleavage (cl. PARP1) was analysed by western blot. mSIN3A served as loading control (n = 3). c HCT116 cells were treated with 1 mM HU and 2 µM MS-275 for 24 h, fixed and incubated with RPA-specific antibody (green). Staining with secondary antibody was performed using Alexa Fluor-488-coupled antibody and TO-PRO3 was used to visualise nuclei. Representative images are shown (n = 3; scale bar, 10 µm). d Analysis of RPA foci per cell in HCT116 cells. Number of foci was determined using ImageJ software. Data represent the mean ± SD (n = 3; one-way ANOVA; **P < 0.01). e HCT116 cells were cultured with HU (1 mM) and/or MS-275 (2 µM) for 24 h. In the following, samples were fixed, incubated with ɣH2AX antibody (phospho-S139-H2AX) and stained with Alexa Fluor-488-coupled secondary antibody (red). TO-PRO3 was used for nuclear staining. Representative images are shown (n = 3; scale bar, 10 µm). f Cells were treated with 1 mM HU and 2 µM MS-275 for 6 and 24 h. Whole-cell lysates were analysed for ɣH2AX by western blot. β-Actin was used as loading control. Numbers indicate densitometric analysis of ɣH2AX signals relative to untreated control and normalised to loading control (n = 3)