Figure 3.

Selection of A2058 clones after high-dose TMZ treatment. (A) Treatment schedule was as follows: 10⁵ A2058 cells (MGMT negative) were seeded into a 10-cm dish and treated with a single dose of 200 µM TMZ for 48 h. Two weeks later, well over a thousand colonies emerged. To enable selection of well-isolated, individual clones, the colonies were pooled and re-seeded at very low density and allowed to form colonies again (in the absence of any further drug treatment). Then, 12 random colonies (numbered 21 through 32) were picked for further analysis by WB and CFA. (B) Six of these clones were subjected to CFA with increasing concentrations of TMZ. Shown is the average IC50 of each clone, in comparison to parental A2058 cells (left bar in black) (n = 2–5, ±SE). (C) Eleven of these cell clones were lysed, and MGMT protein levels were analyzed by WB with actin as the loading control. Left lane shows lysate from parental A2058 cells. Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 21 (right lane, set at 1.0); <0.1 indicates below detection limit.