ACSVL3 knockdown reduced stem cell marker expression and induced differentiation of GBM stem cell enriched neurospheres. A-B. HSR-GBM1A, HSR-GBM1B cells and low passage primary neurosphere cells (JHH612 and JHH626) were incubated with ACSVL3 siRNAs for 72 hours. Total cellular RNAs were extracted and subject to qRT-PCR to detect expression of stem cell markers nestin, sox-2 and Musashi. 18S was used as an internal control for qRT-PCR. ACSVL3 knockdown significantly inhibited stem cell marker expression in GBM stem cell enriched neurospheres. C. ACSVL3 knockdown promoted differentiation of GBM neurosphere cells. GBM neurosphere cells (HSR-GBM1A, HSR-GBM1B) and low passage primary neurosphere cells (JHH612) were transfected with ACSVL3 siRNA for 3 days followed by immunoblotting analysis to detect differentiation markers GFAP (astroglial marker) and Tuj1 (neuronal marker). ACSVL3 knockdown induced a 3-4-fold increase in GFAP expression and a 1.5-2-fold increase in Tuj1 expression, respectively. D. Immunofluorescence staining confirmed the increase of GFAP and Tuj1 expression following ACSVL3 knockdown. Neurospheres cells were collected by cytospin and then stained with anti-GFAP (red) and anti-Tuj1 (green) antibodies. Nuclei were stained with DAPI (blue). ACSVL3 knockdown induced an increase in GFAP and Tuj1 expression.