Expression of Semaphorin 3A and its receptors in endothelial, glioma and glioma stem-like cells. (A) Semaphorin 3A (S3A, green) expression was analysed by confocal microscopy in high grade glioma stem-like cells grown as neurospheres (GSCs#1–#4). Nuclei were counterstained with DAPI (blue). Scale bar: 10 µm. (B–D) S3A expression was checked by RT-PCR (B,D) and by western blotting (C) in GSCs#1–#4, in glioblastoma cell lines (U87, U138, U251, U373 and LN229), SVGp12 foetal glial lines and Jurkat T lymphocytes (Jrk). β-actin and H₂O were used as positive and negative controls, respectively in PCR experiments. Tubulin was used as a protein loading control. (E) Plexin (Plx) A1, A2, A3 and A4 expression was measured by RT-PCR in hCMEC/D3, HUVEC and GSC#1, along with neuropilins (NRP) 1 and 2. (F) S3A expression was evaluated by western blotting in GSC#1 transfected with non-silencing (sic) and three different siRNA sequences targeting S3A. β-catenin (β-cat) was used as a loading control. (G) PlxA1 expression was explored by RT-PCR in non-silencing (sic) and plxA1 siRNA transfected hCMEC/D3. β-actin was used as housekeeping gene. (H) NRP-1 expression was evaluated by western blotting in hCMEC/D3 transfected with non-silencing (sic) and three different siRNA sequences targeting NRP1. Tubulin was used as a loading control.

Glioma stem-like cell-secreted Semaphorin 3A remodels brain endothelial cell monolayers. (A) Three-day-old confluent human brain endothelial cells (ECs) were cultured alone or with glioblastoma stem-like cells (GSCs#1–#4) for 18 h under deprivation conditions. Upper panel: representative phase-contrast images were coloured using Photoshop (ECs in green, GSCs in orange). Repulsion areas are in grey and indicated by black arrows. Scale bar: 60 µm. Lower panel: VE-cadherin (VEC, green) and Sox2 (red) were analysed by confocal microscopy. Scale bar: 10 µm. (B) ECs were cultured with myc–GFP (GFP) and Semaphorin 3A–GFP (S3A)-transfected HEK-293T for 2 days. Representative phase-contrast images were coloured using Photoshop (ECs in green, HEK-293T in blue, repulsion areas in grey). Scale bar: 60 µm. Inserted numbers are the percentage of HEK-293T that repelled EC monolayers; n = 100. (C) Co-culture assays, as described in A, were fixed and processed for immunostaining for Sox2 (red) and VEC or S3A (green). Nuclei were counterstained with DAPI. Scale bars: 10 µm. (D–F) Co-culture assays were performed as described in A. GSCs were either pre-treated for 1 h with pre-immune (ctrl; 2 µg/ml, 1 h) and S3A (N15; 2 µg/ml, 1 h) antibodies (ab) or transfected with 50 nM non-silencing sequence (sic) and S3A siRNA (S3A^si) 3 days earlier. ECs received 50 nM of sic and plexin A1 siRNA (plx^si) 3 days prior exposure to GSCs. Graphs represent the percentage of the total number of GSC neurospheres (NS) that repelled EC monolayers; n = 100.

Semaphorin 3A secreted by glioma stem-like cells jeopardizes brain endothelial cell monolayer integrity. (A,B) Three-day-old confluent human brain endothelial cells (ECs) were exposed for 15 min to glioblastoma stem-like cell conditioned medium (GSC CM) pre-incubated with pre-immune (ctrl, 2 µg/ml, 1 h) and S3A antibodies (ab, N15, 2 µg/ml, 1 h) and to CM prepared from non-silencing (sic)- and S3A siRNA (^si)-transfected GSCs. Alternatively, sic- and plexin siRNA (plx^si)-transfected ECs were incubated with GSC CM. VE-cadherin antibody uptake was performed for each condition. Number of cells exhibiting vesicular staining was quantified and expressed as a percentage of the total number of cells; n = 300. (C) FITC-dextran permeability was measured in hCMEC/D3 treated for 1 h with CM prepared from GSCs#1–#4 transfected with sic and S3A^si. (D) ECs were exposed for 5 min to GSC CM pre-incubated with ctrl- and S3A-blocking antibodies, as defined in B. CM from sic- and S3A^si-transfected GSCs were similarly applied. Phospho-VE-cadherin (p-VEC) and total VEC expression levels were analysed by western blotting.

Semaphorin 3A operates through Src in brain endothelial cell monolayer remodelling. (A) Three-day-old confluent human brain endothelial cells (ECs) were stimulated with 100 ng/ml Semaphorin 3A (S3A) for the indicated times. Phosphorylation (p) levels of Src (Y416) and PP2A-C (Y307) were analysed by western blotting. Src and PP2A-C served as loading controls. Alternatively, PP2A-C immunoprecipitated fractions (IP) were blotted for Src and PP2A. Src and PP2A-C intensity ratios are indicated below. (B) PP2A activity was measured in ECs stimulated with S3A for the indicated times. (C,D) ECs transfected with non silencing (sic) and Src targeting (src^si) duplexes were stimulated with S3A for 5 minutes and analysed for PP2A activity (C) and p-VE-cadherin (p-VEC), Src and VEC by western blotting (D). (E–G) ECs were pre-treated with DMSO vehicle (ctrl) and SU6656 (Src^inh, 1 µM) prior S3A stimulation, and processed for p-VEC and VEC western blotting (E) FITC-dextran passage (F) and VEC antibody uptake (G).

Inhibition of PP2A by Set alters brain endothelial cell monolayer integrity. (A) Mice received intra-dermal injection of DMSO vehicle (ctrl) or PP2A inhibitor (Caly, 1 µM and 100 nM), in duplicate in each flank. The images show the Evans blue extravasation at the site of injection, 1h post-treatment. The graph shows the mean of Evans blue extravasation (n = 6). (B–D) Three day-old confluent human brain endothelial cells (ECs) treated with DMSO vehicle (ctrl) and Caly (10 nM, unless specified) were processed for FITC-dextran passage (B), phospho-VE-cadherin (p-VEC) and VEC western blotting (C), and VEC antibody uptake (D). (E) FLAG immunoprecipitations (IP) were performed in HEK-293T transfected with FLAG–Set and PP2A-Cα, as indicated. FLAG and PP2A-C western blots are shown for IP and total cell lysates (TCL). Additionally, S3A-stimulated ECs were processed for PP2A-C immunoprecipitations (IP). Anti-Set and anti-PP2A-C were monitored by western blotting. (F–H) Non silencing (sic)- and Set-targeting (Set^si)-transfected ECs stimulated with S3A were processed for PP2A activity (F), p-PP2A-C, PP2A-C and Set western blotting (G) and total VEC staining (H). (I–L) Sic- and Set^si-transfected ECs exposed to medium conditioned by glioblastoma stem-like cells (GSC CM) were processed for p-VEC and VEC western blotting (I), VEC antibody uptake (J), FITC-dextran passage (K) and EC monolayer repulsion assays in co-culture (L).