Fig. 8
PR130 influences cell fate decisions. a HCT116ΔgRNA (upper panel) and HCT116ΔPR130 (clone #16; lower panel) cells were incubated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Cells were fixed and incubated with RPA-specific primary and Alexa Fluor-488-coupled secondary antibody. TO-PRO3 was used to visualise nuclei. Representative images are shown; scale bar, 10 µm. b Analysis of RPA foci per cell using ImageJ software. Treatment and staining were performed as described in a. Data presented as mean ± SD (n = 3; two-way ANOVA; **P < 0.01; ***P < 0.001). c Quantification of mean ɣH2AX fluorescence per cell using ImageJ software. At least 50 nuclei per treatment were analysed. Cells were treated with MS-275/HU for 24 h. Results were normalised to untreated controls. Data represent the mean ± SD (n = 3; two-way ANOVA, **P < 0.01). d Western blot analysis of HCT116ΔgRNA and two HCT116ΔPR130 clones after 24 h treatment with 1 mM HU and/or 2 µM MS-275. RAD51 and β-Actin were detected using immunoblot. e Cells were treated with 1 mM HU and 2–5 µM MS-275 for 24 h. Whole-cell lysates were analysed for PARP1 cleavage (cl. PARP1) by western blot. β-Actin served as loading control. f Treatment with HU (1 mM) and/or MS-275 (2 µM) was carried out for 40 h in both HCT116ΔgRNA (black) and HCT116ΔPR130 (clone #3; red). Cell cycle analyses were performed with flow cytometry and representative histograms are shown (n = 3)
