Fig. 6

Effects of NEO212 on MGMT expression and repeated treatments. a, Left panel: TW4 cells were treated with 50, 75, and 100 μM NEO212, 15 μM O6BG (O6), or vehicle (vh.) for 24 h. Control cells received no treatment. Right panel: TW4 cells were treated with 50 μM NEO212 for the indicated time periods. In all cases, cell lysates were prepared and analyzed by Western blot with antibodies specific to MGMT or actin (as a loading control). b, TW4 cells were treated with 1: vehicle only; 2: 50 μM NEO212; 3: 50 μM NEO212 combined with 5 μM MG-132 (a proteasome inhibitor); 4: 5 μM MG-132. After 16 h, cells were harvested and analyzed by Western blot. c, Cells were treated with increasing concentrations of NEO212 at time 0 h (1x Tx). After 24 h, some of the cells received a second dose of the same NEO212 concentration (2x Tx). The number of formed colonies was determined 10 days later. Dotted line marks IC50 values, which shifted to the left upon repeat treatment