Fig. 2

a Decrease of total cell numbers upon different temozolomide (TMZ) treatment strength. LN229 glioma cells were treated with different TMZ concentrations for 10 days, control cells were treated with the solvent DMSO (0.5%). Total cell numbers strongly decreased in a TMZ concentration dependent manner. Given are mean values of cell counting ± SD from n = 3 independent experiments. b Increase of the H2BK positive dormant cell portion upon different TMZ treatment strength, and incorporation of BrdU. The fraction of dormant cells as determined by immunoreactivity for the glioma dormancy marker H2BK and counting of the positively stained cells was remarkably increased in a concentration dependent manner (grey portions of the graph). The fraction of cells with BrdU incorporation in turn decreased (hatched portions of the columns), but, remarkably, in higher TMZ concentrations, H2BK and BrdU double positive cells were frequently observed (hatched, grey portions of the columns). Microscopic pictures exemplarily show cells expressing the dormancy marker H2BK (green) and the incorporation of BrdU (red) upon stimulation with different concentrations of TMZ for 10 days. The pictures are representative examples from 6 to 10 fields of view that were analyzed for n = 4 independent experiments and summarized in the graphs in the upper part; the bars indicate 20 μm. c Influence of cell density on portions of dormant cells. Left: When stained with the vital dye DiO and treated with 100 μg/ml TMZ for 10–12 days, nearly all cells (about 98%) cease from dividing which is indicated by the retention of the green fluorescent dye. Meanwhile, in control treated cells (DMSO), nearly all cells lose the green fluorescent dye due to repeated cell divisions. Right: Graphs show surviving dormant cells after 10 days of TMZ treatment (100 μg/ml) in dependence of the initially seeded cell numbers. Portions of surviving cells are very low in comparison to total (extrapolated) cell numbers in DMSO control cultures, and do not depend on initially seeded cell numbers. Given are mean values of cell counting ± SD from n = 6 independent experiments