t for 5 days significantly decreased cell growth in all lines, with the most rapidly dividing HOG cells most affected. Even though Oli-Neu cells were plated onto PLL-coated dishes, decreased adhesion of these small cells may have contributed to their apparent growth reduction. The effect of GTA-mediated acetate supplementation on ASPA protein levels, acetyl-CoA genesis, and oligodendrocyte differentiation was next assessed. The more rapidly dividing HOG cells possessed more ASPA and AceCS1 protein than Hs683 or Oli-Neu cells. GTA treatment decreased ASPA protein levels in Hs683 and HOG cells, but had no effect on Oli-Neu cells. AceCS1 should be up-regulated to deal with the increased GTA-derived acetate load; however, GTA blunted the temporal increase of AceCS1 in Hs683 and HOG cells and Luteolin 7-glucoside transiently increased AceCS1 protein levels in Oli-Neu cells. No significant regulation of AceCS2 protein levels was observed in the absence or presence of GTA. 
Since ASPA expression is positively correlated with oligodendrocyte differentiation in vivo, it was not surprising that the decreased ASPA levels induced by GTA in Hs683 and HOG cells was associated with decreased CNPase protein levels 5 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g001 . Although GTA 12411425 did not alter ASPA levels in Oli-Neu cells, GTA significantly reduced CNPase protein levels. Since ASPA undergoes cytoplasmic-nuclear shuttling and its co-localization with AceCS1 may play a role in histone acetylation, the spatial distribution of these enzymes was examined. In control cultures, ASPA immunoreactivity was present in both the cytosol and nucleus, but appeared more prominent within the nucleus. Despite its demonstrated role in cytoplasmic lipid synthesis in peripheral tissues, AceCS1 immunoreactivity was predominantly nuclear in all cells and at all time points, similar to the 1685439 expression pattern reported for the developing and adult rat brain. Interestingly, GTA induced cytosolic accumulation of ASPA and a profound morphological alteration in Hs683 cells. GTA appeared to decrease the intensity of CNPase 6 GTA Inhibits Glioma Stem-Like Cell Proliferation doi: 10.1371/journal.pone.0080714.g002 immunoreactivity and the number of immunoreactive cells. Taken together, these data suggest that the promotion of differentiation does not underlie the growth inhibitory effects of GTA on established oligodendroglioma cell lines. GTA promotes growth arrest greater than sodium acetate at comparable acetate concentrations Because GSCs exhibit altered metabolism relative to their non-stem tumor cell counterparts, the growth effect of GTA-derived acetate supplementation was investigated in primary oligodendroglioma-derived cells that exhibit selfrenewal and tumorigenicity. Since the active molecule of GTA is thought to be acetate, the growth inhibitory effects of GTA were compared to 36 mM sodium acetate and 12 mM sodium acetate as a positive control. GTA is cleaved by non-specific lipases and esterases in all cells into acetate and glycerol; thus, the growth effects of 0.25% glycerol alone and with 36 mM sodium acetate also were tested. As an intact molecule, GTA is a short chain triglyceride. There are no naturally occurring short chain triglycerides shorter than palmitate, because it is the first fatty acid produced by fatty acid synthase, and is the precursor for all other longer chain fatty acids. Hence, all shorter triglycerides are synthetic, with GTA being
