Figure 2.
Fibulin-3 is highly expressed in glioma tissue and cells. A) Total homogenates from nine high-grade gliomas (two grade III astrocytomas and seven grade IV glioblastomas) and seven age-matched controls were probed for fibulin-3 (arrow). The asterisk indicates non-specific cross-reactivity of the antibodies used. B) Quantification of results from (A); notice that fibulin-3 expression is virtually absent in normal brain compared to gliomas. I.O.D., integrated optical density. The accompanying western blot shows a representative subcellular fractionation of glioma tissue revealing the distribution of fibulin-3. Nuclear antigen PCNA, microsomal ribophorin and soluble alpha-tubulin were used as specific markers for the nuclei-enriched (N), membrane-enriched (M) and soluble (S) subcellular fractions, respectively. C) Expression of fibulin-3 in the conditioned medium and total lysates of glioma cell lines (U87, U251, U373), primary cultures of glioma xenografts (X12, X14) and cultured normal human astrocytes (NHA). Equal protein loading for culture media was determined by protein measurement and by comparable amido-black staining of the blot membranes prior to incubation with antibody. D) Analysis of relative migration of fibulin-3 from soluble fraction of glioma tissue (tissue, open circle) or medium from U251-MG glioma cells (cells, open triangle) indicated a single form of Mw~54-kDa, as expected. No other fibulin-3 bands were observed. Asterisks indicate antibody cross-reactivity as indicated above. E) Detection of fibulin-3 mRNA in human glioma cell lines, rodent glioma cell lines (rat CNS-1; mouse GL-261 and KR-158) and NHA. The rightmost lane corresponds to RT-PCR in the absence of template. E) Results from qRT-PCR show the comparative expression of fibulin-3 mRNA (mean ± S.E.M.) in NHA (control=1) versus glioma cell lines and glioma neurospheres prepared from patient specimens (OG2, OG6, OG12) as indicated in the methods section. Gliomaspheres cultured in serum-free conditions express fibulin-3 at levels comparable to those of typical glioma cell lines, suggesting that expression of fibulin-3 in culture is not caused by serum-induced mesenchymal drift.