ACSVL3 expression was decreased in differentiated GBM neurosphere cells. A. Western blot analysis of ACSVL3 expression in adherent GBM cells maintained in serum (U373, U87) and in GBM neurosphere cells maintained in serum-free medium [HSR-GBM1A (GBM1A) and HSR-GBM1B (GBM1B)]. Blot was quantified by ImagJ and the fold change over actin is listed underneath. B. qRT-PCR analysis indicated that ACSVL3 mRNA level significantly decreased after GBM neurosphere cells were forced to differentiate (diff) by growth factor withdrawal and 1% serum. Total cellular RNA was extracted from cells 5 days under differentiation conditions. Columns, mean relative ratio of ACSVL3 to 18S RNA from triplicate determinations. C. GBM neurosphere cells (HSR-GBM1A and HSR-GBM1B) were cultured in neurosphere medium (Con) and treated with differentiating agents retinoic acid (RA, 10 μmol/L) or histone deacetylase inhibitor trichostatin A (TSA, 200 nmol/L) for 48 hours. Western blot analysis showed a ∼50-75% decrease in ACSVL3 protein following treatment with the two differentiating agents. Blots were quantified by ImageJ and the average fold changes over actin were listed underneath. D. Western blot analysis for ACSVL3 expression in low passage primary neurosphere cells (JHH626, JHH612 and JHH701) and their differentiated partners (diff) induced by growth factor withdrawal and 1% serum for 5 days. Differentiation resulted in decreased ACSVL3 expression in all three primary GBM neurosphere cultures. E. The expression of another member of the Acyl-CoA synthetase family, ACSF2, was not significantly altered in response to forced differentiation by serum- or RA. F. CD133+ and CD133− cells were isolated from GBM1A neurospheres using microbead-conjugated CD133 antibodies and magnetic columns (Miltenyi Biotec). Messenger RNAs were extracted from the two cell populations and subjected to qRT-PCR. Compared to CD133- cells, CD133+ cells expressed significantly higher levels of ACSVL3 (∼7.5 fold).*: P < 0.05; **: P < 0.01.