BIRC3 impacts GBM cell self-renewal and is dependent on BMP4 suppression. (A). Human BMP4 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (B). Human CD133 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (C). Human ABCG2 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (D). Protein expression of SMAD1, SMAD5 and phosphorylated SMAD1/5 in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. Specific antibodies as indicated. β-actin acts as internal control. (E). Control or BIRC3-KO of U251 cells were seeded in 6 well plates and cultured in neurosphere formation medium. The BIRC3-KO cells had been treated with control siRNA and BMP4 siRNA separately 1 day before seeding. The number of neurospheres were observed and calculated by microscope. Representative images are under 4× magnification (top raw) and 10× magnification (bottom raw). n = 5, * p < 0.05.