Matrigel

Endothelial cord formation was measured by a Matrigel endothelial cord formation assay as described by Wang et al (2013) [42 (link)]. 96 well plates were coated with Matrigel (Corning, Loughborough, UK). Human Umbilical Vein Endothelial Cells HUVECs (Promocell, Heidelberg, Germany) were seeded 1.5×10⁵ cells/well onto Matrigel-coated wells and treated with conditioned media from spheroid cells or non-conditioned control media with or without rhVEGF as positive and negative controls.

HPMECs grown to approximately 80% confluence in 6-well plates were silenced. After 24 h of silencing, the cells were detached with TrypLE Express (Invitrogen, CA, USA), and 1 × 10⁵ cells were added to 250 μl of serum-free Matrigel (PromoCell, Heidelberg, Germany), gently mixed, polarized for 30 min at 37 °C, deposited into a 24-well culture plate (Bio-Rad, Redmond, WA, USA) and treated with 100 pM recombinant human TGFβ1 (R&D Systems, Minneapolis, MN, USA). Cells were then covered with 50 μl of complete medium containing 10% FCS and grown at 37 °C under 5% CO₂ or 90% O₂ for the indicated time periods (0, 4, 12, 24, and 48 h). To inhibit the ALK5 pathway, the cells were treated with 10 μM SB431542, an inhibitor of Smad2 phosphorylation (R&D Systems, Minneapolis, MN, USA). Angiogenesis was evaluated by counting the number of tube and network formations. Only tubular structures connecting two cell clusters were considered for branch measurements. Representative images were taken using a BX51 microscope (Olympus, Tokyo, Japan) with an attached camera (Leica DFC 280; Leica, Wentzler, Germany) at 40 × magnification. At each time point, cells were processed with Dispase (Bio-Rad, Hercules, CA, USA) for biochemical analysis.