*2.3. Cell Culture*

BeWo cells were obtained from the American Type culture Collection (ATCC ® CCL-98 ™) and cultured at 37 ◦C and 95% air 5% CO2 in Hams F12K medium (Cat. N3520, SIGMA-ALDRICH, St. Louis, MO, USA), supplemented with 10% fetal calf serum (Cat. 26140-079, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Cat. 150770, Invitro, S.A., CDMX, Mexico). Cells were plated on Transwell filters (12-well plate, 0.4 μm, Cat. 3460, Corning, Kennebunk, ME, USA); 24 h later, the monolayers were incubated for 1.5 h with ZIKV at MOI = 1. Then, the monolayers were fixed for 10 min with methanol at −20 ◦C for claudin-4 immunofluorescence, or fixed in 4% para-formaldehyde in PBS at RT for 10 min and permeabilized with 0.2% Triton X-100 in PBS for occludin and ZO-1 immunofluorescence.
