Figure 6.

DNER regulates GBM-derived neurosphere cell growth and differentiation and not apoptosis. (A): Neurospheres (line 10627) were dissociated, transfected with control siRNA (SiCon) or with anti-DNER siRNA (SiDNER), and replated in neurosphere growth medium with or without TSA (200 nM) as shown. Seven days later, neurospheres were immobilized in soft agar and those measuring >100 μm diameter per low powered field were quantified by computer-assisted morphometry. Cultures treated with TSA plus SiDNER contained threefold more large neurospheres than cultures treated with TSA alone. DNER expression inhibition rescues neurosphere cells from the growth-inhibiting effects TSA. (B): Neurosphere cells were transfected with control plasmid or with plasmid containing full-length DNER cDNA. Sixty hours later, total cell lysates were assayed for GFAP, TuJ1, and actin by immunoblot analysis. Transgenic DNER induces both GFAP and TuJ1 expression. (C): Neurosphere cells were transfected with control plasmid or with DNER as in (B). Forty-eight hours later, neurospheres and nonadherent cells were collected, dissociated, and subjected to flow cytometry for CD133. DNER expression reduces the fraction of CD133⁺ cells. (D): Neurosphere cells were transfected with TSA with or without SiDNER or transfected with full-length DNER cDNA and then grown under neurosphere growth conditions for 7 days. Nonadherent cells and neurospheres were then collected by cytospin and stained for the differentiation markers GFAP and TuJ1. DNER expression inhibition reversed TSA-induced differentiation and expressing full-length DNER induced differentiation. (E): GBM-derived neurospheres (line 10627) were dissociated, transfected with control siRNA (SiCon) or with anti-DNER siRNA (SiDNER), and replated in neurosphere growth medium with or without TSA (200 nM) as shown. Sixty hours later, neurosphere cell viability was determined by trypan blue exclusion. SiDNER did not alter the fraction of trypan blue-positive cells in either control- or TSA-treated neurospheres. (F): Neurosphere cells were transfected with control plasmid or with plasmid containing full-length DNER cDNA. Sixty hours later, cell viability was determined by trypan blue staining and apoptosis was determined by annexin V flow cytometry. Forced DNER expression had no effect on either parameter of cell viability. Data are shown as the mean ± standard error of the mean (A, C). Bar, 20 μm; *p < .05; **p < .01; NS, p > .05. Abbreviations: Con, control; DNER, Delta/Notch-like epidermal growth factor-related receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBM, glioblastoma; GFAP, glial fibrillary acidic protein; siRNA, small interfering RNA; TSA, trichostatin A.