Characterization of anti-BAI1 antibody and absence of expression of BAI1 in glioma cell lines. A: Expression of BAI1 in normal human brain (from autopsy, 01–89). Equal amounts of lysates (lysed in 8 mol/L urea and 4% SDS) from BAI1 transfected or untransfected U251MG (lanes 1 and 2, respectively), frontal (lane 3), and cerebellar (lane 4) portions of human brain tissue were resolved on a 7% SDS-PAGE and transferred on a nitrocellulose membrane and Western blot analysis was performed with polyclonal-affinity purified anti-BAI1 antibody (upper panel) and with anti-actin (lower panel) antibody. The BAI1-specific 170-kd band observed in both the normal tissue and in the BAI1 transfected cells is indicated. B: The anti-BAI1 antibody does not cross-react with BAI2 and BAI3. BAI1, BAI2, and BAI3 transient expression in 293 cells was achieved by transient transfection using expression plasmids previously described. 1,4 Equal amounts (40 μg) of lysates from human embryonic kidney 293 cells either untransfected (lane 1) or transiently transfected with cDNAs encoding BAI1 (lane 2), BAI2 (lane 3), or BAI3 (lane 4) were resolved on a 7% SDS-PAGE and transferred on a nitrocellulose membrane. Blots were probed for BAI1 expression (upper panel) and actin (lower panel). U251MG cells either untransfected (lane 5) or stably transfected with BAI1 cDNA (lane 6) were used as negative and positive controls, respectively. C: Equal amounts of cell lysates from the indicated cell lines or from normal brain obtained from a temporal (temp) lobectomy of a patient with epilepsy (01–87) were examined for BAI1 (panel 1) or for actin (panel 2) expression by immunoblot analysis. BAI1 mRNA (panel 3) expression was evaluated by RT-PCR analysis using 0.5 of total μg RNA prepared from the indicated human glioma cell lines. Primers and PCR conditions were as described. 1 GAPDH was used as a control for gel loading and mRNA integrity.

Immunohistochemistry of human glioblastoma and non-neoplastic brain specimens. A: Formalin-fixed pellets of U251MG cells untransfected (1) or stably transfected with BAI1 cDNA (2) were used as negative and positive controls for immunohistochemistry. Immunohistochemistry on non-neoplastic human brain tissue from surgically resected specimens shows diffuse expression of BAI1 in the brain parenchyma of both the white matter (3) and cortex (4). Staining is absent in vascular endothelial cells and perivascular stromal cells (arrows). B: Autopsy specimen containing GBM with adjacent non-neoplastic white matter stained by hematoxylin and eosin (1) and by immunohistochemistry for BAI1 (2). Diffuse staining of the brain parenchyma is observed in non-neoplastic brain but not in the adjacent region of GBM (arrowheads). Higher magnifications of the adjacent brain and neoplastic tissue (in boxes in 2) are shown in 3 and 4. C: Examples of GBM resection specimens showing presence (1 and 2) or absence (3) of cytoplasmic BAI1 expression in neoplastic cells by immunohistochemistry. Arrow in 1 points to vessels showing absence of expression of BAI1, and asterisk in 3 points to necrotic areas within the GBM.

BAI1 mRNA is not induced by p53 in glioma cell lines. Induction of BAI1 mRNA by p53 was evaluated by RT-PCR as detailed in the Materials and Methods section. Induction of p21/CDKN1A mRNA by p53 was used as a positive control. Expression of GAPDH mRNA was used as a control for RNA amounts and integrity. A: T98G cells were transiently transfected with wild-type p53 cDNA. The cells were harvested at 12 hours and 24 hours post-transfection and analyzed for BAI1 mRNA induction of expression by p53 using RT-PCR. T98G cells transfected with BAI1 cDNA or mock-transfected cells were used as positive and negative controls, respectively. B: Activation of wild-type p53 activity in GM 47–23 cells by dexamethasone (1 μmol/L for 18 hours). GM 47–23 is a clone derived from T98G glioma cells and contains a wild-type p53 cDNA under a dexamethasone-responsive promoter. T98G parental cells were used as a negative control and T98G transiently transfected with BAI1 cDNA as a positive control. C: Activation of wild-type p53 in 2024 cells by doxycycline 2024 cells contain a wt TP53 gene regulated by doxycycline-responsive promoter and are derived from C16 cells (which are a clone of LNZ308 cells expressing the rtTA transactivator). WT p53 was induced by treating the cells with doxycycline (2 μg/ml) for 24 hours and the cells were then evaluated for BAI1 mRNA induction by RT-PCR. D: LN382 cells harboring a temperature-sensitive p53 mutant were grown at either 37°C or at 34°C (permissive temperature) for 24 hours and analyzed for BAI1 mRNA induction. U251MG cells stably transfected with BAI1 cDNA were used as a positive control.