Fig. 4: Role of RAD18 in the TMZ-induced DDR.
A TMZ induces pATM (S1981) and RAD18 foci. A172 cells were exposed to indicated drugs and RAD18 and pATM (S1981) foci were quantified following immunofluorescent staining. For example left panel, for quantification right panel. Nuclei were stained with TO-PRO-3 (magnification, x630, scale bar, 10 µm). Indicated significances result from an unpaired t-test with Welch’s correction. B siRNA-mediated knockdown of RAD18 sensitizes glioma cells towards TMZ exposure. Following RAD18 knockdown, assessed by western blot analysis (Top), cell death response was determined after TMZ exposure in LN229 and A172 cells (Bottom). As control, cells were transfected with a non-targeting siRNA (siNON). Indicated significances result from a ratio paired t-test. C Overexpressed RAD18 protects glioma cells from entering TMZ-induced cell death. LN229 and A172 cells were exposed to TMZ and transiently transfected with a RAD18-EGFP expressing plasmid. D HDACi prevents TMZ-induced mono-ubiquitination of PCNA as determined by western blot analysis. Glioma cells (LN229, A172) were exposed to indicated drugs. ß-ACTIN served as a loading control. E HDACi sensitizes glioma cells towards the O6MeG lesion induced by TMZ. In MGMT expressing T98G cells (insert, left), MS-275 (1.5 µM) downregulates RAD18 (insert, right) and sensitizes toward TMZ once MGMT is depleted by O6BG (5 µM) (bottom). Cell death represents the sum of apoptosis and necroses. Indicated significances result from an unpaired t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001. For A and D cells were exposed to MS-275 (1.5 µM), TMZ (50 µM) and TMZ/MS-275 and assays were performed 72 h later and 120 h later for E. For B and E the cell death response was determined by flow cytometric analysis of Annexin V-FITC/PI double-stained cells and for C by Sub-G1 analysis 120 h after the exposure to TMZ (50 µM).
