Figure 1.

NTN4 regulates glioblastoma cell proliferation and migration in a concentration-dependent bell-shaped manner. (A) Astrocytes and glioblastoma cell lines express NTN4. Using Q-RT-PCR, NTN4 expression levels were determined in astrocytes and three invasive glioblastoma cell lines: U87MG, U251MG, and U373MG. Glioblastoma cells have a lower expression of NTN4 than astrocytes in vitro. (B and C) U251MG cells were infected by lentivirus harboring NTN4 shRNAs. After puromycin selection for 72 hours, Q-RT-PCR was performed to detect the expression of NTN4. NTN4 shRNAs resulted in NTN4 mRNA reduction of approximately 80% for shRNA1, 95% for shRNA3, and 87% for shRNA4 (B). The silencing effects of these three shRNAs were also detected by immunofluorescence (C). (D–F) Cells expressing NTN4 shRNAs were used for proliferation assay, migration assay, and apoptosis assay. Mitogenic rate, migration distance, and survival rate of NTN4-silenced cells were compared with nontransfected cells (wt) or cells transfected with nontargeting shRNA (Scr). In the apoptosis assay, cells cultured in 10% serum-containing medium were used as positive control. Suppression of NTN4 gene expression in U251MG cells significantly reduced cell mitogenicity, migration, and increased apoptosis caused by serum deprivation. (G and H) U251MG cells were cultured in serum-free medium for 4 hours and then cultured in the presence of recombinant NTN4 with the indicated concentrations. Recombinant NTN4 was reapplied every 24 hours for 48 hours. Cells were then used for proliferation assay. NTN4 promoted U251MG cell proliferation at 50 and 200 ng/ml, but it inhibited cell proliferation at 3 µg/ml (G). U251MG cells were cultured in serum-free medium overnight and then used for the wound closure assays in the presence of recombinant NTN4 as in the indicated concentration. NTN4 promoted U251MG cell migration at 50 and 200 ng/ml. However, 5 µg/ml NTN4 had no significant effect on U251MG cell migration (H). Mean ± SE, n ≥ 3. **P < .01. *P < .05.