Figure 1. Undifferentiated THP-1 cells accumulate HIF-1α in response to hypoxia and LPS.

(A) THP-1 cells were incubated under normoxic (NOX) and hypoxic (HOX) conditions for up to 16 h in the presence or absence of LPS (1μg/ml). A 70 μg portion of total cell lysate was resolved by SDS/PAGE. HIF-1α protein was detected with an anti-HIF-1α antibody, and anti-α-tubulin antibody was used to demonstrate equal gel loading. (B) THP-1 cells were incubated for 3 h with LPS. Total RNA was prepared, reverse-transcribed into cDNA, and HIF-1α cDNA was quantified by real-time PCR. Amounts of HIF-1α cDNA were normalized to 1 μg of total RNA. Shown are the means±S.D. from four separate experiments, *P<0.05. (C) LPS and hypoxia increase HIF-1 DNA-binding activity. An 80 μg portion of nuclear extract from treated THP-1 cells was incubated with a biotin-labelled double stranded oligonucleotide containing a specific HIF-1 binding sequence or a mutated (mut) HRE for 30 min at room temperature. Detection of DNA-bound HIF-1 complexes was performed using streptavidin–HRP conjugate. Binding activity of HIF-1 from LPS- and hypoxia-treated cells were calculated as a percentage of that exhibited by untreated normoxic controls. n=3, ***P<0.001. (D) LPS induces the expression of the HIF-1 target gene. THP-1 cells were incubated under normoxic and hypoxic condtions in the presence and absence of LPS (1 μg/ml) for 6 h. Total RNA was isolated and reverse-transcribed into cDNA. ADM cDNA was quantifed by real-time PCR. Shown are the means±S.D. from four separate experiments; *P<0.05.