ACSVL3 knockdown depleted GBM neurosphere cells expressing stem cell markers. A. ACSVL3 expression was knocked down with two ACSVL3 siRNAs: ACSVL3 si3 and ACSVL3 si4. Quantitative RT-PCR (qRT-PCR) analysis indicated that compared to scrambled siRNA transfected cells (con), ACSVL3 mRNA level decreased ∼55-60% following ACSVL3 siRNA transfection for 72 hours in HSR-GBM1B cells. 18S was used as an internal control for qRT-PCR. Columns, mean relative ratio of ACSVL3 to 18S RNA from triplicate determinations. B-D. GBM neurosphere cells (HSR-GBM1A and HSR-GBM1B) were transiently transfected with ACSVL3 siRNAs and cultured in serum-free medium. Neurosphere cells were dissociated and subjected to flow cytometry to detect CD133-expressing cells (B) and ALDH-expressing cells (D). Total cellular proteins from neurospheres were collected and subjected to immunoblotting analysis for CD133 expression (C). ACSVL3 down-regulation significantly decreased the percentage of neurosphere cells with CD133+ and ALDH+. *: P < 0.05; **: P < 0.01.