4.3. Co-Culture of Vero Cells and HUVECs or Vero Cells and BEAS2B Cells

For co-culturing of Vero cells and HUVECs or Vero cells and BEAS2B cells, Vero cells were seeded in the inserts of 24-well plates (0.4 µm polyester membrane, Costar, Thermo Fisher Scientific) while HUVECs or BEAS2B cells were grown in wells without inserts. After 48 h, the insert with Vero cells were transferred to the well of HUVECs or BEAS2B cells. Cells were then infected by adding SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 to the inserts. The co-cultures were kept in co-culture media (HUVECs and Vero cells co-culture medium was ½ HUVEC medium and ½ Vero cell medium; Vero cells and BEAS2B cells co-culture medium was Vero cell medium). After 72 h, media were removed and cells were fixed with 10% buffered formalin. For the EPAC agonist or antagonist treatment, the co-culture media for Vero cells and HUVECs were replaced with 5 µM I942 or NY0173 [42]-supplemented media at 24 h p.i. At 72 h p.i., media were removed and cells were fixed with 10% buffered formalin. Dimethyl sulfoxide (DMSO)-supplemented medium was used as the vehicle. All experiments were performed in replicates of three or more.