Figure 3.

Many GBM cell lines are addicted to TANK. (a) A172 cells were retrovirally transduced to express short hairpin RNAs (shRNAs) leading to a knockdown of TANK, TBK1 or a scrambled shRNA as a control. Transduced cells were selected with puromycin and used to determine cell proliferation (upper) or alternatively protein expression and cell viability (lower). Proliferation was measured by plating out equal numbers of cells, followed by determination of cell numbers after 3 and 5 days using a FACSCalibur. Error bars show standard deviations from three different experiments. Aliquots of the cells were lysed and tested by western blotting for efficient knockdown. Equal numbers of cells were also seeded and used 2 days later for MTT assays, which reflect the metabolic activities and cell proliferation rates. Relative metabolic activity of control cells was arbitrarily set as 1, error bars show standard deviations. (b and c) The experiment was done as in (a) but it shows the experiment for cells, which are representative for groups that are only partially addicted from TBK1 (b) or TBK1 and TANK (c). (d) Summary of the cell proliferation assays. ++ shows completely absent and + shows impaired proliferation after knockdown. ND indicates not determinable, as TBK1 knockdown in T98G and U118 cells turned out not to be efficient.