Quantifying Temozolomide Intracellular Levels

For the experiment, cells were seeded in T75 flasks (Sarstedt AG & CO). When the confluence of the flasks was around 75%, cells were pre-treated with honokiol (35 µM for U373 and 40 µM for Hs683), MOMIPP (3 µM), vacquinol-1 (5 µM), or left untreated for different periods of time (3, 15 and 22 h) before the addition of TMZ (200 µM) for 2 h. After the treatment, the culture medium was removed, the cells were washed twice with cold PBS (Gibco)), scrapped in 200 µL of ice-cold methanol (VWR International, Oud-Heverlee, Belgium), and sonicated for 30 s. As TMZ is stable at pH < 4 [80 (link)], 50 µL of cell lysate was diluted in 50 µL of an acid internal standard solution (2 µM theophyline and 0.1% formic acid in methanol).