Global differential DNA methylation phenotypes in response to zebularine concentration. Maximum intensity projections (MIP) of 3D-imaged nuclei of DU145 cells with high similarity values within their population category are selected for display (bars are 5 μm). Gradual increase in reduction of MeC sites (false-colored green) was observed in correlation with an increase in zebularine concentration, similarly in both cell types. At lower concentrations (8 and 40 μM) global demethylation seems to be preferentially stronger at the nuclear periphery (delineated by DAPI, false-colored blue) and less DAPI-dense areas (supposedly euchromatin), and gradually affects more interior regions with increased drug concentrations. At higher drug doses (200 μM and especially 500–1000 μM), also more DAPI-dense areas (heterochromatin) have been demethylated. This latter effect is even more pronounced in the AZA-treated cell nuclei (data not shown in here). However, the majority of heterochromatic regions seem to retain their compact conformation. The respective scatter plots of the nuclei provide more quantitative information regarding changes in the MeC/DAPI codistribution as a consequence of drug application, especially for the lower drug doses: a demethylation of non-heterochromatic sites (MeC-positive, low-DAPI signals) is indicated for 40 μM compared to 8 μM, as judged by the decline of the graph slope. This trend correlates with increasing drug concentration. At 200–1000 μM the leveling of the slope towards the x-axis becomes obvious; additionally also strong DAPI-positive sites (heterochromatin) have started to become hypomethylated.