HIF-α induction by BiPS. Quiescent VSMC, quiescent BAEC, or HeLa cells were maintained under control conditions, in hypoxic conditions (1% O₂), in the presence of CoCl₂ (200 μM), or various concentrations of BiPS or vehicle (DMSO 0.08%) for 2 h. Total cell extracts (25 μg) were resolved by SDS-PAGE (8%) and immunoblotted using anti-HIF-1α, anti-HIF-2α, or anti-p42/p44 MAPK antibodies.

Time course for HIF-α induction by BiPS. HeLa cells were maintained under control conditions, in hypoxic conditions (1% O₂), or in the presence of 75 μM BiPS or vehicle (DMSO 0.08%) for different periods of time. Total cell extracts (25 μg) were resolved by SDS-PAGE (8%) and immunoblotted using anti-HIF-1α, anti-HIF-2α, or anti-p42/p44 MAPK antibodies.

Stabilization of HIF-1α by BiPS. A, HeLa cells were transfected with 0.5 μg of CMV-luc-HIF-1α-ODDD and 250 ng of an expression vector coding for R. reniformis luciferase. At 40 h after transfection, cells were maintained under control conditions, hypoxic conditions (1% O₂), or in the presence of BiPS (75 μM) or MG132 (5 μM) for 6 h. Cells were lysed, and luciferase activity was measured. Results are expressed as a ratio of beetle luciferase activity to R. reniformis luciferase activity and are an average ± S.E.M. of at least three independent experiments performed in triplicate. B, cytoplasmic extracts from HeLa cells treated as indicated were incubated with HIF-1α (344–582) GST protein-coupled Sepharose beads. Samples were then incubated overnight in the presence of in vitro translated pVHL and resolved by SDS-PAGE (12%). Immunoblotting was performed using anti-HA (pVHL) and anti-GST antibodies. C, HeLA cells were treated with MG132 (25 μM) for 2 h and followed by the addition of CoCl₂ (200 μM) or BiPS (75 μM) for an additional 2 h. Total cell extracts (25 μg) were resolved by SDS-PAGE (8%) and immunoblotted using anti-hydroxylated Pro402 (P402-OH), anti-hydroxylated Pro564 (P564-OH), anti-HIF-1α, or anti-p42/p44 MAPK antibodies.

Modulation of PHD activity by BiPS. A, FeCl₂ (20 μM) was incubated with BiPS (75 μM) or deferoxamine (50 μM) for 30 min. FerroZine (20 mg/ml) and ammonium acetate (1 mg/ml) were then added to the reaction mixture and incubated at 37°C for 30 min. Results are expressed as total free iron as determined by comparison with a FeCl₂/FerroZine standard curve and are an average ± S.E.M. of three independent experiments. B, cytoplasmic extracts from HeLa cells were supplemented with dimethyl-2-oxoglutarate (33 μM) and/or treated with BiPS (100 μM) for 20 min before incubation with HIF-1α (344–582) GST protein-coupled Sepharose beads. Samples were then incubated overnight in the presence of in vitro translated pVHL and resolved by SDS-PAGE (12%). Immunoblotting was performed using anti-HA (pVHL) and anti-GST antibodies.

Increased HIF-1 nuclear complex formation by BiPS. HeLa cells were maintained under control conditions, in hypoxic conditions (1% O₂), or in the presence of BiPS (75 μM) for 2 h. Nuclear protein extract (10 μg) was incubated in a 96-well plate coated with an oligonucleotide containing the wild-type (W26) or mutant (M26) HIF-1-binding site. The presence of HIF-1 transcription complex was evaluated using anti-HIF-1α and anti-HIF-1β antibodies. Results are expressed as the fold increase of absorbance at 450 nm over control conditions and are an average ± S.E.M. of at least three independent experiments performed in triplicate.

HIF-dependent reporter gene activation by BiPS. HeLa cells (six-well plate) were transfected with 1 μg of a pGL3 (R2.2) 3HRE-TK reporter construct and 250 ng of an expression vector coding for R. reniformis luciferase to normalize transfection efficiency. At 40 h after transfection, cells were maintained under control conditions, in hypoxic conditions (1% O₂), or in the presence of BiPS (75 μM) or vehicle (DMSO 0.08%) for 6 h. Cells were lysed, and luciferase activity was measured. Results are expressed as a ratio of beetle luciferase activity to R. reniformis luciferase activity and are an average ± S.E.M. of at least three independent experiments performed in triplicate.

HIF-dependent VEGF expression by BiPS. BAECs were transfected with 20 nM siRNA oligonucleotides targeting HIF-1α, HIF-2α, or with a control siRNA. At 40 h after transfection, cells were maintained under control conditions, in hypoxic conditions (1% O₂), or in the presence of BiPS (75 μM) or vehicle (DMSO 0.08%) for 2 h. Top, total RNA was extracted and resolved on formaldehyde/agarose gels. Northern blot was performed using a specific radiola-beled VEGF probe. An 18S RNA probe was used as a control for gel loading. Bottom, total cell extracts (25 μg) were resolved on SDS-PAGE (8%) and immunoblotted using anti-HIF-1α and anti-HIF-2α antibodies.