Anti il 1α

DENV2 strain 16881 was propagated in C6/36 Aedes albopictus cells and titrated by plaque assay, as previously described (36 (link)). Supernatant from uninfected cell cultures produced in the same conditions was used as negative control (Mock). Platelets from healthy volunteers were incubated in M199 with DENV2 in a multiplicity of infection of 1 virus per platelet (MOI = 1) or incubated with the same amount of Mock for 6 hours at 37°C. After stimuli, samples were centrifuged at 900 X g for 15 minutes for supernatant harvesting and cell preparation for flow cytometry or Western blot. To investigate the mechanisms involved in platelet iNOS expression and NO production, platelets were incubated with DENV-2 in the presence or absence of IL-1 receptor antagonist (IL-1RA), anti-IL-1α (R&D 840201) or anti-IL-1β (R&D 840168) or isotype-matched control antibody.

For immunofluorescence staining, ECs were grown on gelatine-coated coverslips in standard culture 12-well plates. At confluency, culture medium was removed and cells were rinsed twice with pre-warmed HBRS. Subsequently, different stimuli (UBC cell SN alone or in combination with inhibitors (anti IL-1α 20 ng/ml (R&D systems, Wiesbaden, Germany); anti-IL-1β 30 ng/ml (R&D systems, Wiesbaden, Germany); anti-IL1ra 320 ng/ml (R&D systems, Wiesbaden, Germany)) were added to the cells at indicated concentrations. Pre-warmed starvation medium served as a control. The SN of endothelial cells were collected after 6 h of incubation at 37 °C, centrifuged at 300 g for 5 min to clear detritus, and kept at − 20 °C for later enzyme-linked immunosorbent assay (ELISA) analysis. The cells on coverslips were fixed and subjected to immunofluorescence staining. For qRT-PCR or flow cytometry, HUVECs were grown to confluence in standard 10 cm petri dishes or 25 cm² flasks, then medium was removed, and cells were rinsed twice with pre-warmed HBRS. Subsequently, T24 cell SN or starvation medium (control) was added and incubated for 12 h.

Lysates, cytosolic and nuclear extracts were electrophoresed on a 4-12% polyacrylamide gel (Invitrogen) and proteins were transferred to an Immobilon membrane (Millipore). Membranes were blotted with anti-IκBα (Cell Signaling, #4814), anti-IκBβ (R&D systems, AF5225), anti-p50 (Abcam, ab7971), anti-p65 (Cell Signaling, #8242), anti-IL-1α (R&D systems, AF-400-NA), anti-HDAC (for nuclear extracts, Cell Signaling, #5356), anti-Calnexin (for lysate, Enzo Life Sciences ADI-SPA-860), and anti-GAPDH (for cytosolic extracts, Cell Signaling, #5174). Densitometric analysis was performed using Image Studio (LiCor).