Calpain 2

VSMCs were lysed in RIPA buffer. Protein concentrations were quantified with protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated overnight with primary antibodies against HIF-1α (1:1000, Abcam), calpastatin, TGF-β1 (1:2000, Abcam), calpain-1, calpain-2, a-II spectrin (1:3000, Abcam), MMP2, TIMP2 (1:400, Boster), MT1MMP (1:200, Boster), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, Cell Signaling Technology). At room temperature, membranes were incubated with corresponding secondary antibodies for 1 hour. Quantitative analysis was performed with Image J software (NIH). All samples were run in triplicate and averaged.

Gli4 and GliT cultures were fixed by 4% PFA. Cell and brain sections were blocked and permeabilized using PBS - triton 0.5% - horse serum 5%. Primary antibodies were incubated overnight at 4 °C. The antibodies used in immunofluorescence were: N-Cadherin (abcam ab12221), Calpain-2 (abcam ab155666), Vinculin (Sigma Aldrich V9264), actin cytoskeleton was stained with Alexa Fluor 488 phalloidin (Molecular Probes A12379), cell nuclei with Hoechst 33342 (H3570 Life technologies) and Human Nuclei (Millipore MAB1281). Fluorochrome-coupled secondary antibodies were incubated 2 hours at room temperature (dilution 1/500). Image acquisition was realized using Zeiss Axioimager Z1/ Zen (with an apotome). Z-stack acquisition was realized using confocal microscopy (LSM 700). Imaris 8.1.2 software was used for image 3D reconstitution.
Migration quantifications were done using ZEN 2012 software in counting number of cells, using dapi staining, from 200 µm away from the border of the NS to the last observed migrating cell. Alternatively, migration capacity was quantified by measuring an area of migration. To measure migration areas, we subtracted the area of the NS containing non-migrating cells from the total area where cells were detected.

For western blot analysis, cells were lysed with ice-cold RIPA buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics). Lysates were clarified by centrifugation at 13000 rpm for 30 min, after which the supernatants were harvested. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo). Equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking nonspecific binding sites on the membranes with 5% skim milk or 5% BSA for 2 hours at RT, the membranes were incubated with the indicated primary antibodies overnight at 4°C and then with the appropriate secondary antibodies for 1 hour at RT. Antibodies against Cyclin B, Cyclin D1, E2F1, survivin, PARP, EGFR, FAK, Src, α-tubulin, GAPDH (Cell Signaling Technology), TfR1, calpain-1, calpain-2 (Abcam), c-Met (Invitrogen), and c-MYC (Santa Cruz Biotechnology) were obtained commercially. Immunoreactive bands were visualized with HRP-conjugated secondary antibodies and a chemiluminescent substrate by exposure to X-ray film.