Medium 200prf

A transwell system was used for examining the interaction between HCC cell lines and HUVECs. The lower compartment of each 24-well transwell plate was coated with Matrigel. Each well was seeded with 4×10⁴ HUVECs in Medium 200PRF (Thermo Fisher, Waltham, MA, USA). The upper compartment of each transwell plate was seeded with 1×10⁴ HuH7-S2, HuH7-core-high, or HuH7-core-low cells in serum-free DMEM. The upper compartment was then placed onto the lower compartment and the transwell plates were incubated at 37°C in a 5% CO₂ environment for 6 hours. Calcein AM fluorescent dye was added and incubation was continued for another 30 minutes. Next, the cells in the lower compartment were examined under a microscope and quantitatively analyzed using MetaMorph (Molecular Devices, Sunnyvale, CA, USA). For validating the effects of VEGF, 1 mg/mL bevacizumab (Roche, Switzerland) or control immunoglobulin G1 (IgG1) was added to the culture media of both the HCC cells and HUVECs. For validating the effects of AP-1 inhibition, 50 μM of an AP-1 inhibitor, T-5224 (Apexbio, Houston, TX, USA), was added to the culture media of both the HCC cells and HUVECs.

Human umbilical vein endothelial cells (HUVECs, purchased from ATCC) were cultured and maintained in Medium 200PRF supplemented with low serum growth supplement (Thermo Fisher Scientific). For the tube formation assay, HUVECs were labeled with Calcein AM dye (Thermo Fisher Scientific), and mixed with Geltrex matrix (Thermo Fisher Scientific) to a final concentration of 5 × 10⁴ cells/mL. The mixture was plated onto a 96-well μ-Plate (ibidi GmbH, Germany). Each well was cultured in either MSTC-conditioned or control (unsupplemented) medium, collected as described above in the Sample Collection section, and imaged 16 h later. The endothelial tubes were traced and quantified using Fiji and the AngioTool plugin.^{13 (link),14 (link)}