measured in 60 tumor cell lines by dielectrophoresis. For comparison, we also analyzed the plasma membrane folding in two non-malignant human cell lines, including the human embryonic kidney HEK293 line 
and the human fibroblast cell line HFIB-1. As evident from the Fig. S3, the mean Cm values of 1.5660.10 and 2.0560.12 mF/cm2 obtained, respectively, for HEK293 and HFIB-1 cells, are similar to that of DK-MG cells, i.e. the lowest Cm value among 5 tested GBM lines. To verify our conclusion that the extremely PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19650784 high isotonic Cm values of GBM cells, were really due to excessive membrane folding, we analyzed the impact of hypotonic cell swelling on Cm. The rationale of these experiments was to prove whether, and to what extent, the hypotonic stretching and expected membrane unfolding would cause a reduction of Cm as commonly found in mammalian cells. Excessive Membrane Folding in Glioblastoma Cells In agreement with previous studies, our ROT experiments revealed a strong dependence of Cm on the external osmolality in all tested GBM lines. Thus, decreasing the osmolality from 300 to 50 mOsm caused the Cm of DK-MG cells to decrease from the isotonic Cm = 1.88 mF/cm2 to the hypotonic Cmh = 1.05 mF/cm2. In case of U373-MG cells, the same osmotic shift resulted in a much greater absolute and percentage reduction of Cm from 4.0 to 1.19 mF/cm2. In general, the hypotonically stressed cells from different GBM lines showed similar Cmh values lying within a narrow range between 0.97 and 1.19 mF/cm2, even though the isotonic Cm data were widely different. Being somewhat larger than the flat-membrane Cm0 = 0.8 mF/cm2, the hypotonic Cmh values of GBM cells suggest that, despite severe hypotonicity, the cell surface was not perfectly smooth. The remaining folds/microvilli can be explained by the large original membrane excess in GBM cells present under isotonic conditions. Unlike Cm, the whole-cell DHMEQ web capacitance CC of GBM cells did not show any significant changes despite considerable cell swelling in hypotonic medium, as evident from the comparison of isotonic cell radius and CC values in Osmotic Properties of Glioblastoma Cells The consistently low Cmh values obtained for all GBM lines in strongly hypotonic medium suggest that the microvilli and membrane folds, rather than other mechanisms, were responsible for the very high isotonic Cm values. These membrane extensions are well known to provide an instantaneous source of material to preserve the plasma membrane integrity during hypotonic swelling. Given the large excess of Excessive Membrane Folding in Glioblastoma Cells the imposed osmotic gradient. The rates and magnitudes of initial swelling varied widely among the cell lines. DK-MG cells swelled somewhat slower than other cell lines, which is particularly evident from the data obtained in 50- and 100-mOsm sucrose solutions. The Vmax values obtained in sucrose media were used to evaluate the osmotically inactive volume fractions b of cells by applying the Boyle van’t Hoff equation. As seen in despite persisting hypotonicity. RVD relies on the release of cytosolic solutes through swelling-activated membrane pathways. In agreement with our findings presented here and previously, other glioma cells are able to readjust their volume in anisotonic media. In sharp contrast to the disaccharide sucrose, the small organic osmolyte inositol not only completely abolished RVD, e.g. in case of DK-MG cells, but also caused noticeable secondary swelling of GaMG and SNB