Ldev free reduced growth factor basement membrane matrix

The cellular migration ability was measured by a transwell assay using chambers with transwell inserts of 8 µm in pore size in a 24-well plate. 5×10⁴ cells suspended in 200 µL normal culture medium were seeded in the upper chamber for HUVECs with shNC (negative control shRNA), shRhoJ (RhoJ targeting shRNA), shRhoJ+con (shRhoJ combined with plenti-control), or shRhoJ+oe (shRhoJ combined with plenti-RhoJ), medium 200 supplemented with 20 ng/ml VEGF was placed in the lower chamber. For the U87 and U251 transwell assay, the conditioned medium (CM) of HUVECs-shNC or HUVECs-shRhoJ was placed in the lower chamber. 24 h after seeding, cells were fixed with 4% PFA and stained with 1% crystal violet staining solution.
The angiogenesis ability of HUVECs was conducted by in vitro tube formation assay. LDEV-Free Reduced Growth Factor Basement Membrane Matrix (A14132-02, Gibco, USA) was thawed at 4 ℃ overnight. The next day, Matrix was placed on a pre-chilled 24-well plate and then coagulated at 37 ℃ for 30 min. 1×10⁵ cells/well were seeded in the Matrix coated plate and cultured for 16-18 h, then the cells were fixed and photographed under a light microscope.

Geltrex LDEV-free reduced growth factor basement membrane matrix (Gibco, Burlington, ON, Canada, cat# A1413201) was used. The Geltrex basement membrane matrix was thawed overnight in a 4 °C fridge. Once thawed, it was mixed well and aliquoted (80 μL/well) into a 96-well plate (on ice) with chilled pipette tips. To prevent air bubble formation, the basement membrane was dispensed without a full stop, and the plate was centrifuged at 300× g for 10 min at 4 °C. Then, the coated plate was incubated at 37 °C and 5% CO₂ for 30 min. HUVECs were cultured and transfected as described above. The cells were serum-starved for 4 h and detached with trypsin-EDTA, then resuspended at 2 × 10⁵ cells/mL in complete EGM-2 medium. Each coated well received 100 μL of cell suspension (2 × 10⁴ cells/well). Tube formation was carried out at 37 °C under an atmosphere of 5% CO₂ and live-imaged with a Hamamatsu Camera and a Zeiss Axio Observer microscope (5× magnification) with Zen Pro 3.6 software. The images were processed at the 16 h time point with Zen 3.3 Lite (Blue Edition) and analyzed with Angiogenesis Analyzer 1.0 software for ImageJ2 or Fiji. All conditions were done in duplicate.

Spheroids were generated using the liquid overlay technique. 2000 cells/well (in 100 µL medium) were seeded into U-bottom 96-well plates, precoated with a thin layer of 1.5% low gelling agarose (Sigma Aldrich, St. Louis, MO, USA). LNCaP and T-47D were cultured scaffold-free, whereas 3% Geltrex^TM (LDEV-Free Reduced Growth Factor Basement Membrane Matrix, Gibco, Paisley, UK) was added to the PC-3 cell suspension, followed by centrifugation at 161× g for 10 min at 4 °C. During spheroid establishment, any influence of Geltrex^TM on radiosensitivity was excluded (see Supplementary Figure S1).