Figure 1. Cytotoxicity end points upon siRNA-mediated XAF1 silencing in malignant glioma cells exposed to TMZ.

XAF1 knockdown was performed 24 h prior to the treatment of the cells. Time points indicated refer to the treatment time (A) Flow cytometric analysis of apoptosis induction (subG1) is shown in LN229 cells after XAF1 knockdown (XAF1-si) and transfection with non-coding siRNA (con-si). Cells were treated with 100 μM TMZ and fixed after the time points indicated. After PI staining, the subG1 fraction was determined. Error bars indicate the SD in three independent experiments in duplicates (N = 3). Data were analyzed for statistically significant differences by two-tailed t-test, comparing target XAF1-si vs. con-si. A p-value of < 0.05 was considered to be statistically significant (*). The efficiency of siRNA-mediated XAF1 knockdown was verified by western blot analysis 24 h after transfection. HSP90 was used as loading control. (B) Flow cytometric analysis of annexin V-FITC/PI - stained LN229 cells upon transfection with con-si and XAF1-si RNA and exposure to 100 μM TMZ for 96 and 120 h. The corresponding unexposed controls (con 96 h, con 120 h) are shown. Error bars indicate the SD in two independent experiments in duplicates (N = 2). Data were analyzed for statistically significant differences by two-tailed t-test, comparing target XAF1-si vs. con-si. A p-value of < 0.01 was considered to be statistically significant (**). (C) The cell viability (metabolic competence) upon treatment with 100 μM TMZ was determined by MTT assay in con-si and XAF1-si transfected LN229 cells. Technical triplicates at 96 and 120 h are shown. (D) Colony forming assay of LN229 cells transfected with con-si and XAF1-si RNA, exposed to increasing TMZ concentrations (semi-logarithmic scale). Two experiments in duplicates are shown. The unexposed controls were set to 100%.