Fig. 5. β8 integrin regulates VM formation and invasive phenotype of GBM cells via activating TGFβ1/p-smad3/RhoA signaling.

A Levels of secreted TGFβ1 in culture medium of GSC#1 and GSC#2. B Migration abilities of GBM cells co-cultured with β8⁺ GSCs were assessed with the addition of neutralizing antibody targeting TGFβ1 or TGFβ receptor inhibitor LY2109761. C Tube formation capacities of GBM cells treated in B were assessed. D Statistical analysis of migration and tube formation assay. E Protein levels of CDH5, N-Cadherin and Vimentin in G4 cells were measured via immunoblotting. F Co-IP of ITGB8 and LAP (TGFβ1) in GSC#s analyzed by immunoblotting. G Confocal images of immunofluorescence staining for ITGB8 (red), LAP (green) and dapi (blue) in GSC#2 revealed co-localization of ITGB8 and LAP. Scale bar = 50 μm. H Six GBM cells were untreated or treated with 100 pM TGFβ1 for 6 h, indicated protein levels were determined by immunoblotting. I Immunofluorescence staining of p-Smad3 and ROCK1 in G4-shTGFβ1 cells co-cultured with β8⁺ or β8⁻ GSC#2. J GBM cells G4-shTGFβ1 and A172-shTGFβ1 were treated with SIS3 (3 μM) and Y27632 (10 μM) for 1 h, followed by co-culturing with β8⁺ GSC#2 for 12 h. Migration and tube formation capacities were then determined. K Statistical analysis of migration and tube formation assay. L G4-shTGFβ1 was treated under same condition described above. Levels of p-Smad3, Smad2/3, ROCK1, CDH5, N-Cadherin and Vimentin were measured via immunoblotting. M G4-shTGFβ1 cells were pretreated with SIS3 (3 μM) or Y27632 (10 μM) for 1 h and subsequently co-cultured with β8⁺ or β8⁻ GSC#2 respectively. Immunofluorescence staining for F-actin was conducted with phalloidin. Scale bar = 50 μm. N Filopodia quantitation in G4-shTGFβ1 was measured and statistically analyzed. Uncropped western blot images are shown in Supplementary Fig. 6. Results are represented as mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant.