HIF-1 mediates hypoxia-induced inflammation.

<p>(A and B) Microglia were treated with 3 µM 2ME2, 1 µM YC-1, or the indicated combinations for 6 hours. The protein levels of IL-8 and TNF-α were determined. (C) Detection of IL-8 and TNF-α by ELISA in microglia transfected with siRNA Con or siRNA HIF-1α (24 h). Experiments performed in triplicate showed consistent results. Data are presented as the mean±SD of three independent experiments. * <i>P</i><0.05.</p

Hypoxia induced cell death and inflammation of microglia cells.

<p>(A) To assess cell death in vitro, microglia with the treatment for the indicated time were subjected to PI staining, and analyzed by flow cytometry. The percentage of cells with PI-positive relative to total cell number at each treatment is shown. (B) The effect of hypoxia on the viability of microglia cells. Microglia was treated with the indicated time. Cell viability was assessed using MTT. (C, D, E and F) microglia were treated with the indicated time. The mRNA and protein levels of IL-8 and TNF-α were determined. Experiments performed in triplicate showed consistent results. Data are presented as the mean ±SD of three independent experiments. * <i>P</i><0.05.</p

HIF-1α mediates microglia autophagy induced by hypoxia.

<p>(A) Microglia cells were transfected with siRNAs targeting HIF-1α (100 nM each) for 24 hrs following hypoxia treatment for 16 hrs, and then the protein levels of the target were evaluated by western blot. (B and C) Confocal microscopy and electron microscopy detected autophagosomes. (D) Microglia cells were transfected with siRNA HIF-1α, after treated with hypoxia for 16 hrs. Flow cytometry detected acridine orange positive cell. The asterisks denote significant differences from controls (* <i>P</i><0.05). Experiments performed in triplicate showed consistent results.</p

HIF-1 mediates hypoxia-induced cell death.

<p>(A) Microglia cells were treated with hypoxia for 0, 2, 4, 16, 24 and 48 h. The kinetics of HIF-1α induction was assayed by qRT-PCR. (B) Measurement of HIF-1α in microglia with the indicated treatments by western blot assay. (C and D) microglia were treated with 3 µM 2ME2, 1 µM YC-1, or the indicated combinations for 6 hours, and the cells were cultured with hypoxia for 16 hrs. The percentage of dead cells was determined using the MTT assay or cell death assay. (E and F) Detection of the inhibition efficiency of siRNAs against HIF-1α. Microglia cells were transfected with siRNAs targeting HIF-1α (100 nM each) for 24 h, and the protein and RNA levels of the target was evaluated by western blot and q-PCR assays. (G and H) The effect of cell death on hypoxia transfected with siRNA control and HIF-1α in microglia (24 h). The % PI positive cells have been normalized to 100% in the control. Experiments performed in triplicate showed consistent results. Data are presented as the mean ± SD of three independent experiments. * <i>P</i><0.05.</p

Autophagy is induced in microglia after hypoxia treatment.

<p>(A) Hypoxia induced complete autophagic flux in microglia. The cells were treated with hypoxia for the indicated time in the presence of Baf A1 (10 nM). Control represents the normal oxygen conditions. (B) Microglia cells were transfected with a plasmid expressing GFP-MAP1LC3B. After 24 hrs, the cells were exposed to hypoxia for 6 hrs. Cells were visualised by confocal microscopy immediately after fixation. The number of GFP-MAP1LC3B puncta in each cell was counted. (C) Ultrastructural changes in hypoxia-treated microglia. The control shows samples without hypoxia treatment. Closed arrows indicate autophagosomes. (D and E) Microglia were treated with hypoxia for 6 hrs and stained with 1 mg/ml acridine orange or 50 mM MDC for 15 min. After incubation, cells were immediately analysed by flow cytometry. The bar chart demonstrates an increase in mean fluorescent intensity. The asterisks denote significant differences from controls (* <i>P</i><0.05). Experiments performed in triplicate showed consistent results.</p