Fig 1.
Highly schematic diagram indicating the location of subependymomys (SEs) in the human brain.
The main findings on AQP distributions are also indicated.
Table 1.
Patient history.
Table 2.
Primary antibodies used in human subependymomas.
Table 3.
Primer sequences and product sizes.
Fig 2.
Immunofluorescent double staining for aquaporins in SEs.
Double staining of GFAP- (green) and AQP4 (red) in infratentorial (A, patient 5) and supratentorial (B patient 6) SE. Dapi stained nuclei in blue. Double staining of AQP4 (red) and AQP1 (green) in infratentorial (C patient 4) and supratentorial (D patient 6) SE. AQP 1 and AQP4 positive cells are more or less evenly distributed in infratentorial tumors, in contrast they were found only in the border region of supratentorial tissue. Bar: 50μm (A, B, C); 100μm (D).
Fig 3.
Overview immunhistological staining of AQP4- and -1 in SE.
Infratentorial SE tissue shows AQP4 and -1 immunoreactivity over the whole tumor (A patient 5, C patient 4), whereas supratentorial SE shows AQP4 and -1 staining almost always at the natural border regions of the tumor (B patient 6, D patient 8). Bar 1000μm.
Fig 4.
PCR analysis of AQP4 and AQP1 infratentorial (patients 1–5 or lane 1–5 respectively) and supratentorial (patients 6–10 or lane 6–10) respectively SE.
A: In every location AQP4 (Exon 4–5) was expressed. Lane 11: negative control (H2O), lane 12 positive control (lung). HPRT lane 1–12. B: In every location AQP1 was expressed. The infratentorial SE samples showed very distinct bands in the gel, whereas the AQP1 expression in supratentorial SEs varied and where less distinct. Lane 11: negative control (H2O), lane 12 and 13 positive control (Normal Brain). HPRT1 lane 1–13.
Fig 5.
AQP4 and AQP1 expression (normalized to HPRT1) in SE and normal brain.
A: AQP4 was upregulated in both SE subgroups (ΔCp -4.7 to -6.75) compared to normal brain (ΔCp -1.06/-1.09) (B). (ΔCp-values <-1 mean upregulation, >1 mean downregulation). B: Infratentorial SE show upregulated AQP1 expression (ΔCp -1.27 to -4.49) in contrast to supratentorial SE (ΔCp 1,83 to -1.44) and Normal Brain (ΔCp 1.81/2.48), which show a slight downregulation (A). Blue: patients 1–5 infratentorial SE, red: patients 6–10 supratentorial SE, green: Normal Brain as positive controls.
Fig 6.
Freeze fracture electron micrograph.
A Replica from tissue of patient 5: SE (infratentorial), no OAPs were found, B: Healthy astrocytic endfoot membrane of rat cortex is covered with OAPs. Bar 250 nm.
Fig 7.
Immunhistological staining of agrin (A-D) and alpha-dystroglycan (E-H) in SE.
Neither infratentorial SE (A, C, E, G, patient 5) nor supratentorial SE (B, D, F, G, patient 9) were positive for agrin or dystroglycan, normally found around the blood vessels (for positive control see supplement S3 Fig Bar 1000 and 50μm.
Fig 8.
Staining for MMPs in SE tissues.
Infratentorial (A, C, E, patient 5) and supratentorial (B, D, F, patient 9) SE tissues were stained with antibodies against MMP2 (A, B), MMP3 (C, D), and MMP9 (E, F). The inserts with double letters show a higher magnification as indicated. None of the immunostains showed significant immunoreactivity. For controls, see S3 Fig.
Table 4.
Summary of results.