Figure 4.

MGMT protein levels in mass cultures after TMZ treatment. (A) Treatment schedule was as follows: 10⁵ cells were seeded into the wells of a 6-well dish and treated with a single dose of TMZ at increasing concentrations from 0 to 500 µM for 48 h. Surviving cells were kept in culture until the population had regrown sufficiently to harvest enough cells for WB analysis. (B) Cell lysates were prepared from mass cultures of surviving A2058 cells and analyzed by WB for MGMT protein levels. Actin was used as a loading control. A lysate of A375 cells was used as a positive control for MGMT protein. (C) The same experiment was also performed with M24 cells. Please note that MGMT protein was undetectable before TMZ treatment in both cell lines. Numbers under the blots indicate quantification of MGMT bands, with reference to the actin signal, and relative to the highest TMZ concentration used (right lane, set at 1.0); <0.1 indicates below detection limit.