Ecm625

For CM collection, irradiated glioma cells (0 and 20 Gy) were cultured in Endothelial Cell Basal Medium (EBM, Lonza) for 1 day, then harvested and filtered through 0.2 μm filters (16534, Sartorius Stedim Biotech, Goettingen, Germany). We performed tubule formation assays using the in vitro angiogenesis assay kit (ECM625, Chemicon, Temecula, CA, USA) according to manufacturer instructions. Briefly, HRECs (7 × 10³ cells/well) were seeded on Matrigel in EGM-2, EBM, or CM from irradiated or non-irradiated cells (0 and 20 Gy) at 37 °C for 5 h. Three random view fields per well were examined, and the tubes were counted.

The angiogenesis assay kit was purchased from Chemicon (ECM625). First, 50 µl of ECM matrix gel solution was applied to each well of a 96-well plate. The plate was then incubated at 37 ℃ for 1 h to allow the matrix solution to solidify. Next, 150 µl of endothelial cell growth medium (EGM) was added to each well of the 96-well plate. HUVECs at passage 5 were then seeded onto the surface of the polymerized ECM matrix at a density of 1 × 10⁴ cells per well followed by the experimental conditions as follows: NT, 20 µg/ml of HSA NPs, 5 µg/ml of bFGF protein, and 10 µg/ml of HSA-bFGF NPs (containing 2.5 µg/ml of bFGF protein). The experiment was repeated five times (n = 5). At designated time after incubation with protein or protein nanoparticles (0, 2, 4, 6, and 8 h), the wells were examined to analyze the cell sprouting under a 40X magnification using a Nikon microscope. At the extended time (9, 18, and 24 h), the observation was conducted to reveal the intercellular connections and stability at a 10X magnification with the same Nikon microscope (Fig. S5).

HUVECs were seeded at a density of 5 × 10⁴ cells per well in a 48‐well plate precoated with 150 µL of EC‐Matrigel per well (catalog no. ECM625; Chemicon). Tube formation was quantified 6 h after various treatments or siRNA transfections by analyzing the sprouting tube‐like structures in five randomly selected fields at 40 × magnification using an inverted phase‐contrast microscope.