Human umbilical vein endothelial cells (HUVECs) were prepared by a previously described method [29 (link)]. The HTNV strain 76–118 and inactivated HTNV (mock virus) control were prepared and stored in our lab as described [7 (link)]. For all infections, the virus was allowed to adsorb to HUVECs at a multiplicity of infection (MOI) of approximately 1 in serum-free EGM maintenance medium for 2 h at 37°C. The cells were then washed and incubated in EGM growth medium with 10% fetal bovine serum. The proportion of infected HUVECs was tested using immunofluorescence. At 48 hours postinfection, over 90% of the HUVECs expressed viral nucleocapsid protein in the cytoplasm.
HUVECs were seeded 24 h before treatment. When the cell confluence up to 60%-70%, the cells were infected with HTNV/mock virus (MOI = 1) for 48 h or treated with interleukin-33 (IL-33, R&D system, USA) for 6 h; alternatively, the cells were pre-infected with HTNV/mock for 48 h and then treated with 20 ng/ml IL-33 for another 6 h. To assess the role of ST2 and its signalling pathways in the induction of pro-inflammatory cytokines via IL-33 stimulation, HUVECs were exposed to human recombinant sST2 (R&D Systems, USA) or inhibitors (Calbiochem, USA) of the indicated signalling pathways at different concentrations for 2 h prior to the stimulation indicated above.
HUVECs were seeded 24 h before treatment. When the cell confluence up to 60%-70%, the cells were infected with HTNV/mock virus (MOI = 1) for 48 h or treated with interleukin-33 (IL-33, R&D system, USA) for 6 h; alternatively, the cells were pre-infected with HTNV/mock for 48 h and then treated with 20 ng/ml IL-33 for another 6 h. To assess the role of ST2 and its signalling pathways in the induction of pro-inflammatory cytokines via IL-33 stimulation, HUVECs were exposed to human recombinant sST2 (R&D Systems, USA) or inhibitors (Calbiochem, USA) of the indicated signalling pathways at different concentrations for 2 h prior to the stimulation indicated above.