Figure 2
Hypoxia induces translocation of high-mobility group box 1 (HMGB1) via reaction oxygen species (ROS) production in normal human nasal epithelium (NHNE) cells. (a) NHNE cells that were incubated under hypoxic conditions in the presence or absence of N-acetyl cysteine (NAC) treatment were stained with dichlorofluorescin diacetate (DCFDA) for 20 min at 37 °C and stained cells were visualized by confocal microscopy. (b) Mean fluorescence was quantified using Image J software. Results are shown as mean±s.e.m. (N=3). *P<0.05. (c) NHNE cells were incubated under hypoxic conditions in the presence or absence of NAC pretreatment and an immunofluorescence assay was performed for HMGB1 (green) and 4',6-diamidino-2-phenylindole (DAPI) staining (blue) for DNA. (d) Culture supernatants from NHNE cells were collected and concentrated. A western blot assay was performed, to compare the levels of HMGB1 protein secreted into the extracellular space after hypoxia with/without NAC pretreatment. (e) Enzyme-linked immnunosorbent assay (ELISA assay) using apical NHNE culture supernatant was performed to compare the levels of HMGB1 protein secreted into the extracellular space after hypoxia with/without NAC pretreatment. (N=3). *P<0.05.
