DV induces translocation of HMGB1 from cell nuclei to cytoplasm and into the extracellular milieu.

<p>A, Immunofluorescence analyses (IFA) were performed on DV-infected K562. K562 cells were infected at M.O.I. 10 and fixed at 3 d.p.i. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. B, K562 cells were mock-infected, treated with UV-irradiated DV (UV-DV) at a M.O.I. 10, infected with DV (M.O.I. 10), or treated with 5 µg/ml of LPS. The nuclear and cytosolic fractions were harvested at 3 d.p.i. and Western blot analysis was performed to detect for HMGB1. The band intensities of HMGB1 of the mock-infected, UV-DV treated, DV-infected and LPS-stimulated nuclear fractions were expressed as a ratio to its respective cytosolic fraction. The housekeeping proteins TFIID and actin were included as loading controls for the nuclear and cytosolic fractions, respectively. C, K562 cells were infected with DV at M.O.I. of 1. Cell culture supernatants from DV-infected K562 were harvested at 3 d.p.i. and concentrated for the detection of HMGB1 via Western blot. D, DV-infected PBM cells infected at M.O.I. 1 and the cells fixed at 3 d.p.i. for IFA. HMGB1 was stained green with rabbit anti-HMGB1 antibody and goat anti-rabbit IgG FITC conjugated antibody while DV was stained red using mouse anti-E antibody and anti-mouse-IgG Alexa Fluor 532. The cell nuclei were stained blue with DAPI. E, PBM were infected with DV at a M.O.I. of 1 and Western blot analysis of HMGB1 in the cell culture supernatants of DV-infected PBM was performed. The band intensities of HMGB1 from DV-infected sample and LPS-treated cells at day 1 p.i. were assigned to a value of 1. The relative fold difference in the intensities of DV-infected samples and LPS-treated cells from day 2- to 5 p.i. was measured in relation to the band intensity its respective treatment group at day 1 p.i.