We also thank Dr. in the Assisting Information. Electrocompetent cells of the HIF-1 RTHS were prepared and transformed with the C+5 SICLOPPS plasmid library. Transformation efficiency, assessed by plating 10-collapse serial dilutions of the recovery answer on LB agar supplemented with chloramphenicol (35 g/mL), was consistently found to be 5 107, therefore ensuring adequate protection of the 3.2 106 member cyclic peptide library. Transformants were washed with minimal press and plated onto minimal press agar plates supplemented with ampicillin (50 g/mL), spectinomycin (25 g/mL), kanamycin (50 g/mL), 3-AT (7.5 M), IPTG (100 M), l-arabinose (6.5 M), and chloramphenicol (35 g/mL). The plates were incubated for 2C3 days at 37 C until individual colonies were visible. Colonies were picked and restreaked onto LB agar plates comprising ampicillin (50 g/mL), spectinomycin (25 g/mL), and chloramphenicol (35 g/mL) and incubated over night at 37 C. Surviving colonies from these plates were cultivated over night and assessed by drop-spotting 10-collapse serial dilutions onto minimal press plates, supplemented with antibiotics, IPTG and 3-AT as above, with and without 6.5 M l-arabinose. Plasmids from strains showing a growth advantage in the current presence of arabinose had been isolated and retransformed in to the first selection stress and reassessed for IPTG-dependent inhibition of development, and arabinose development recovery. SICLOPPS plasmids from colonies demonstrating the anticipated phenotypes had been assessed because of their HIF-1 specificity by change into two similar RTHS, aside from the substitute of HIF-1 with unrelated proteins (ATIC, a homodimeric enzyme found in purine biosynthesis, and P6/UEV, a heterodimeric relationship necessary for the budding of HIV from contaminated cells).24,25 Plasmids that caused a growth-advantage in the P6/UEV or ATIC RTHS were discarded for being non-specific. The activity from the cyclic peptides encoded by the rest of the SICLOPPS plasmids was positioned by retransforming in to the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identification of the adjustable insert locations encoding the energetic cyclic peptides was uncovered by DNA sequencing. Peptide Synthesis Cyclic peptides were characterized and synthesized seeing that detailed in the Helping Details. HIF Luciferase Reporter Assays Endogenous HIF-1 luciferase reporter assays were conducted as previously reported in MCF-7 and U2OS-HRE-luc15 cells.46 For plasmid-expressed HIF- luciferase reporter assays, MCF-7 and U2OS cells were transfected with plasmids expressing HIF-1 transiently, HIF-2, or a empty control (pcDNA3.1-HIF-1, pcDNA3.1-HIF-2, or pcDNA3.1), a renilla-encoding control (phRL-TK), and a HIF-dependent firefly luciferase reporter build (pGL2-TK-HRE), using Transfast (Promega) based on the producers guidelines. After 24 h, cells had been retrieved and plated (4000 cells/well) in 96-well plates (Perkin-Elmer) and incubated for 5 h before either hypoxic or aerobic incubation in existence or lack of cyclic peptide inhibitors. Firefly and renilla actions had been motivated using Dual-Glo Reagent (Promega) based on the producers guidelines. The luciferase sign was normalized using the matching renilla beliefs. Recombinant Creation of HIF-1 and HIF-1 HIF-1, HIF-2, HIF-1, bHLH, PAS-A, PAS-B, and PAS-B had been portrayed in (BL21.DE3) seeing that detailed in the Helping Details. In Vitro Assays Draw downs, ELISA, fluorescent binding assays, and ITC had been conducted as complete in the Helping Details. Dosing Cells with Inhibitors Cells had been treated using the mentioned concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia for 4 h, accompanied by incubation within a hypoxic environment. All manipulation of cell pellets (e.g., lysis, mRNA, and proteins removal) was executed within a hypoxic environment. Duolink Closeness Ligation Assay Duolink closeness ligation assay was executed using the in situ PLA Package (O-Link Bioscience, Uppsala, Sweden) based on the producers guidelines. The antibodies utilized had been rabbit monoclonal anti-HIF-1 (NB100-449, Novus Biologicals) and mouse monoclonal anti-HIF-1 (H00000405-B01P, Abnova). Cells were dosed with inhibitors seeing that incubated and over within a hypoxic environment for. Electrocompetent cells from the HIF-1 RTHS had been changed and ready using the C+5 SICLOPPS plasmid library. RTHS, and SICLOPPS collection had been constructed as comprehensive in the Helping Details. Electrocompetent cells from the HIF-1 RTHS had been prepared and changed using the C+5 SICLOPPS plasmid collection. Transformation efficiency, evaluated by plating 10-flip serial dilutions from the recovery option on LB agar supplemented with chloramphenicol (35 g/mL), was regularly found to become 5 107, hence ensuring adequate insurance coverage from the 3.2 106 member cyclic peptide collection. Transformants had been washed with reduced mass media and plated onto minimal mass media agar plates supplemented with ampicillin (50 g/mL), spectinomycin (25 g/mL), kanamycin (50 g/mL), 3-AT (7.5 M), IPTG (100 M), l-arabinose (6.5 M), and chloramphenicol (35 g/mL). The plates had been incubated for 2C3 times at 37 C until specific colonies had been visible. Colonies had been selected and restreaked onto LB agar plates including ampicillin (50 g/mL), spectinomycin (25 g/mL), and chloramphenicol (35 g/mL) and incubated over night at 37 C. Making it through colonies from these plates had been grown over night and evaluated by drop-spotting 10-collapse serial dilutions onto minimal press plates, supplemented with antibiotics, IPTG and 3-AT as above, with and without 6.5 M l-arabinose. Plasmids from strains displaying a growth benefit in the current presence of arabinose had been isolated and retransformed in to the unique selection stress and reassessed for IPTG-dependent inhibition of development, and arabinose development save. SICLOPPS plasmids from colonies demonstrating the anticipated phenotypes had been assessed for his or her HIF-1 specificity by change into two similar RTHS, aside from the alternative of HIF-1 with unrelated proteins (ATIC, a homodimeric enzyme found in purine biosynthesis, and P6/UEV, a heterodimeric discussion necessary for the budding of HIV from contaminated cells).24,25 Plasmids that triggered a growth-advantage in the ATIC or P6/UEV RTHS had been discarded to be nonspecific. The experience from the cyclic peptides encoded by the rest of the SICLOPPS plasmids was rated by retransforming in to the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identification of the adjustable insert areas encoding the energetic cyclic peptides was exposed by DNA sequencing. Peptide Synthesis Cyclic peptides had been synthesized and characterized as complete in the Assisting Info. HIF Luciferase Reporter Assays Endogenous HIF-1 luciferase reporter assays had been carried out as previously reported in U2OS-HRE-luc15 and MCF-7 cells.46 For plasmid-expressed HIF- luciferase reporter assays, MCF-7 and U2OS cells were transiently transfected with plasmids expressing HIF-1, HIF-2, or a empty control (pcDNA3.1-HIF-1, pcDNA3.1-HIF-2, or pcDNA3.1), a renilla-encoding control (phRL-TK), and a HIF-dependent firefly luciferase reporter build (pGL2-TK-HRE), using Transfast (Promega) based on the producers guidelines. After 24 h, cells had been retrieved and plated (4000 cells/well) in 96-well plates (Perkin-Elmer) and IL8RA incubated for 5 h before either hypoxic or aerobic incubation in existence or lack of cyclic peptide inhibitors. Firefly and renilla actions had been established using Dual-Glo Reagent (Promega) based on the producers guidelines. The luciferase sign was normalized using the related renilla ideals. Recombinant Creation of HIF-1 and HIF-1 HIF-1, HIF-2, HIF-1, bHLH, PAS-A, PAS-B, and PAS-B Polidocanol had been indicated in (BL21.DE3) while detailed in the Helping Info. In Vitro Assays Draw downs, ELISA, fluorescent binding assays, and ITC had been conducted as complete in the Assisting Info. Dosing Cells with Inhibitors Cells had been treated using the mentioned concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia for 4 h, accompanied by incubation inside a hypoxic environment. All manipulation of cell pellets (e.g., lysis, mRNA, and proteins removal) was carried out inside a hypoxic environment. Duolink Closeness Ligation Assay Duolink closeness ligation assay was carried out using the in situ PLA Package (O-Link Bioscience, Uppsala, Sweden) based on the producers Polidocanol guidelines. The antibodies utilized had been rabbit monoclonal anti-HIF-1 (NB100-449, Novus Biologicals) and mouse monoclonal anti-HIF-1 (H00000405-B01P, Abnova). Cells had been dosed with inhibitors as incubated and above inside a hypoxic environment for 4 h, after which these were set with cool methanol for 10 min and permeabilized with 0.2% Triton (diluted in PBS) for 10 min. After preincubation using the Duolink Blocking Reagent for 1 h, examples had been incubated over night with the principal antibodies to HIF-1 (1:500) and HIF-1 (1:500). Duolink PLA reagents and probes were added while recommended from the makes guidelines. Quantitative Reverse-Transcription Polymerase String Response Total RNA was extracted from MCF-7 and U2Operating-system cells using RNeasy Mini Package (74104, QIAGEN) and quantified utilizing a Nanodrop ND-1000 spectrophotometer. Complementary DNA was synthesized inside a 20 L response including 1 g of total.We are grateful to Prof. Margaret Ashcroft for U2OS-HRE-luc and U2OS-luc control cells. had been cultured and manipulated (DNA, RNA, and proteins extraction) inside a H35 hypoxia workstation (Don Whitley Scientific) in 1% O2, 5% CO2 and 94% N2. Luminecence was assessed inside a GloMAX-96 microplate luminometer (Promega). All assays had been carried out in triplicate. Data was examined in Excel (Microsoft) or Prism (GraphPad Software program). SICLOPPS Testing for HIF-1 Dimerization Inhibitors The HIF-1 RTHS, connected control RTHS, and SICLOPPS collection had been constructed as comprehensive in the Helping Details. Electrocompetent cells from the HIF-1 RTHS had been prepared Polidocanol and changed using the C+5 SICLOPPS plasmid collection. Transformation efficiency, evaluated by plating 10-flip serial dilutions from the recovery alternative on LB agar supplemented with chloramphenicol (35 g/mL), was regularly found to become 5 107, hence ensuring adequate insurance from the 3.2 106 member cyclic peptide collection. Transformants had been washed with reduced mass media and plated onto minimal mass media agar plates supplemented with ampicillin (50 g/mL), spectinomycin (25 g/mL), kanamycin (50 g/mL), 3-AT (7.5 M), IPTG (100 M), l-arabinose (6.5 M), and chloramphenicol (35 g/mL). The plates had been incubated for 2C3 times at 37 C until specific colonies had been visible. Colonies had been selected and restreaked onto LB agar plates filled with ampicillin (50 g/mL), spectinomycin (25 g/mL), and chloramphenicol (35 g/mL) and incubated right away at 37 C. Making it through colonies from these plates had been grown right away and evaluated by drop-spotting 10-flip serial dilutions onto minimal mass media plates, supplemented with antibiotics, IPTG and 3-AT as above, with and without 6.5 M l-arabinose. Plasmids from strains displaying a growth benefit in the current presence of arabinose had been isolated and retransformed in to the primary selection stress and reassessed for IPTG-dependent inhibition of development, and arabinose development recovery. SICLOPPS plasmids from colonies demonstrating the anticipated phenotypes had been assessed because of their HIF-1 specificity by change into two similar RTHS, aside from the substitute of HIF-1 with unrelated proteins (ATIC, a homodimeric enzyme found in purine biosynthesis, and P6/UEV, a heterodimeric connections necessary for the budding of HIV from contaminated cells).24,25 Plasmids that triggered a growth-advantage in the ATIC or P6/UEV RTHS had been discarded to be nonspecific. The experience from the cyclic peptides encoded by the rest of the SICLOPPS plasmids was positioned by retransforming in to the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identification of the adjustable insert locations encoding the energetic cyclic peptides was uncovered by DNA sequencing. Peptide Synthesis Cyclic peptides had been synthesized and characterized as complete in the Helping Details. HIF Luciferase Reporter Assays Endogenous HIF-1 luciferase reporter assays had been executed as previously reported in U2OS-HRE-luc15 and MCF-7 cells.46 For plasmid-expressed HIF- luciferase reporter assays, MCF-7 and U2OS cells were transiently transfected with plasmids expressing HIF-1, HIF-2, or a empty control (pcDNA3.1-HIF-1, pcDNA3.1-HIF-2, or pcDNA3.1), a renilla-encoding control (phRL-TK), and a HIF-dependent firefly luciferase reporter build (pGL2-TK-HRE), using Transfast (Promega) based on the producers guidelines. After 24 h, cells had been retrieved and plated (4000 cells/well) in 96-well plates (Perkin-Elmer) and incubated for 5 h before either hypoxic or aerobic incubation in existence or lack of cyclic peptide inhibitors. Firefly and renilla actions had been driven using Dual-Glo Reagent (Promega) based on the producers guidelines. The luciferase sign was normalized using the matching renilla beliefs. Recombinant Creation of HIF-1 and HIF-1 HIF-1, HIF-2, HIF-1, bHLH, PAS-A, PAS-B, and PAS-B had been portrayed Polidocanol in (BL21.DE3) seeing that detailed in the Helping Details. In Vitro Assays Draw downs, ELISA, fluorescent binding assays, and ITC had been conducted as complete in the Helping Details. Dosing Cells with Inhibitors Cells had been treated using the mentioned concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia for 4 h, accompanied by incubation within a hypoxic environment. All manipulation of cell pellets (e.g., lysis, mRNA, and proteins removal) was executed within a hypoxic environment. Duolink Closeness Ligation Assay Duolink closeness ligation assay was executed Polidocanol using the in situ PLA Package (O-Link Bioscience, Uppsala, Sweden) based on the producers guidelines. The antibodies utilized had been rabbit monoclonal anti-HIF-1 (NB100-449, Novus Biologicals) and mouse monoclonal anti-HIF-1 (H00000405-B01P, Abnova). Cells had been dosed with inhibitors as above and incubated within a hypoxic environment for 4 h, and they were set with frosty methanol for 10 min and permeabilized with 0.2% Triton (diluted in PBS) for 10 min. After preincubation using the Duolink Blocking Reagent for 1 h, examples had been incubated right away with the principal antibodies to HIF-1 (1:500) and HIF-1 (1:500). Duolink PLA probes and reagents had been added as suggested with the companies guidelines. Quantitative Reverse-Transcription Polymerase String Response Total RNA was extracted from MCF-7 and U2Operating-system cells using RNeasy Mini Package (74104, QIAGEN) and quantified utilizing a Nanodrop ND-1000 spectrophotometer. Complementary DNA was synthesized within a 20 L response filled with.We are grateful to Prof. Margaret Ashcroft for U2OS-HRE-luc and U2OS-luc control cells. cell lines had been preserved in DMEM (Life Technologies) made up of 10% FBS; for aerobic incubation, cells were cultured at 37 C in 5% CO2. For hypoxic treatment, cells were cultured and manipulated (DNA, RNA, and protein extraction) in a H35 hypoxia workstation (Don Whitley Scientific) in 1% O2, 5% CO2 and 94% N2. Luminecence was measured in a GloMAX-96 microplate luminometer (Promega). All assays were conducted in triplicate. Data was analyzed in Excel (Microsoft) or Prism (GraphPad Software). SICLOPPS Screening for HIF-1 Dimerization Inhibitors The HIF-1 RTHS, associated control RTHS, and SICLOPPS library were constructed as detailed in the Supporting Information. Electrocompetent cells of the HIF-1 RTHS were prepared and transformed with the C+5 SICLOPPS plasmid library. Transformation efficiency, assessed by plating 10-fold serial dilutions of the recovery answer on LB agar supplemented with chloramphenicol (35 g/mL), was consistently found to be 5 107, thus ensuring adequate protection of the 3.2 106 member cyclic peptide library. Transformants were washed with minimal media and plated onto minimal media agar plates supplemented with ampicillin (50 g/mL), spectinomycin (25 g/mL), kanamycin (50 g/mL), 3-AT (7.5 M), IPTG (100 M), l-arabinose (6.5 M), and chloramphenicol (35 g/mL). The plates were incubated for 2C3 days at 37 C until individual colonies were visible. Colonies were picked and restreaked onto LB agar plates made up of ampicillin (50 g/mL), spectinomycin (25 g/mL), and chloramphenicol (35 g/mL) and incubated overnight at 37 C. Surviving colonies from these plates were grown overnight and assessed by drop-spotting 10-fold serial dilutions onto minimal media plates, supplemented with antibiotics, IPTG and 3-AT as above, with and without 6.5 M l-arabinose. Plasmids from strains showing a growth advantage in the presence of arabinose were isolated and retransformed into the initial selection strain and reassessed for IPTG-dependent inhibition of growth, and arabinose growth rescue. SICLOPPS plasmids from colonies demonstrating the expected phenotypes were assessed for their HIF-1 specificity by transformation into two identical RTHS, except for the replacement of HIF-1 with unrelated proteins (ATIC, a homodimeric enzyme used in purine biosynthesis, and P6/UEV, a heterodimeric conversation required for the budding of HIV from infected cells).24,25 Plasmids that caused a growth-advantage in the ATIC or P6/UEV RTHS were discarded for being nonspecific. The activity of the cyclic peptides encoded by the remaining SICLOPPS plasmids was ranked by retransforming into the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identity of the variable insert regions encoding the active cyclic peptides was revealed by DNA sequencing. Peptide Synthesis Cyclic peptides were synthesized and characterized as detailed in the Supporting Information. HIF Luciferase Reporter Assays Endogenous HIF-1 luciferase reporter assays were conducted as previously reported in U2OS-HRE-luc15 and MCF-7 cells.46 For plasmid-expressed HIF- luciferase reporter assays, MCF-7 and U2OS cells were transiently transfected with plasmids expressing HIF-1, HIF-2, or a blank control (pcDNA3.1-HIF-1, pcDNA3.1-HIF-2, or pcDNA3.1), a renilla-encoding control (phRL-TK), and a HIF-dependent firefly luciferase reporter construct (pGL2-TK-HRE), using Transfast (Promega) according to the manufacturers instructions. After 24 h, cells were recovered and plated (4000 cells/well) in 96-well plates (Perkin-Elmer) and incubated for 5 h before either hypoxic or aerobic incubation in presence or absence of cyclic peptide inhibitors. Firefly and renilla activities were decided using Dual-Glo Reagent (Promega) according to the manufacturers instructions. The luciferase signal was normalized using the corresponding renilla values. Recombinant Production of HIF-1 and HIF-1 HIF-1, HIF-2, HIF-1, bHLH, PAS-A, PAS-B, and PAS-B were expressed in (BL21.DE3) as detailed in the Supporting Information. In Vitro Assays Pull downs, ELISA, fluorescent binding assays, and ITC were conducted as detailed in the Supporting Information. Dosing Cells with Inhibitors Cells were treated with the stated concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia for 4 h, followed by incubation in a hypoxic environment. All manipulation of cell pellets (e.g., lysis, mRNA, and protein extraction) was conducted in a hypoxic environment. Duolink Proximity Ligation Assay Duolink proximity ligation assay was conducted using the in situ PLA Kit (O-Link Bioscience, Uppsala, Sweden) according to the manufacturers instructions. The antibodies used were rabbit monoclonal anti-HIF-1 (NB100-449, Novus Biologicals) and mouse monoclonal anti-HIF-1 (H00000405-B01P, Abnova). Cells were dosed with inhibitors as above and incubated in a hypoxic environment for 4 h, after which they were fixed with chilly methanol for 10 min and permeabilized with 0.2% Triton (diluted in PBS) for 10 min. After preincubation with the Duolink Blocking Reagent for 1 h, samples were incubated overnight with the primary antibodies to HIF-1 (1:500) and HIF-1 (1:500). Duolink PLA probes and reagents were added as recommended by the.Josephine Corsi for helpful discussions and technical assistance. Supporting Information Available Supporting Information includes Figures S1CS9, Tables S1 and S2, and supplemental methods. This material is available free of charge via the Internet at http://pubs.acs.org Author Present Address ? P.A.T.: Institute for Cancer Sciences, University of Manchester, St. RNA, and protein extraction) in a H35 hypoxia workstation (Don Whitley Scientific) in 1% O2, 5% CO2 and 94% N2. Luminecence was measured in a GloMAX-96 microplate luminometer (Promega). All assays were conducted in triplicate. Data was analyzed in Excel (Microsoft) or Prism (GraphPad Software). SICLOPPS Screening for HIF-1 Dimerization Inhibitors The HIF-1 RTHS, associated control RTHS, and SICLOPPS library were constructed as detailed in the Supporting Information. Electrocompetent cells of the HIF-1 RTHS were prepared and transformed with the C+5 SICLOPPS plasmid library. Transformation efficiency, assessed by plating 10-fold serial dilutions of the recovery solution on LB agar supplemented with chloramphenicol (35 g/mL), was consistently found to be 5 107, thus ensuring adequate coverage of the 3.2 106 member cyclic peptide library. Transformants were washed with minimal media and plated onto minimal media agar plates supplemented with ampicillin (50 g/mL), spectinomycin (25 g/mL), kanamycin (50 g/mL), 3-AT (7.5 M), IPTG (100 M), l-arabinose (6.5 M), and chloramphenicol (35 g/mL). The plates were incubated for 2C3 days at 37 C until individual colonies were visible. Colonies were picked and restreaked onto LB agar plates containing ampicillin (50 g/mL), spectinomycin (25 g/mL), and chloramphenicol (35 g/mL) and incubated overnight at 37 C. Surviving colonies from these plates were grown overnight and assessed by drop-spotting 10-fold serial dilutions onto minimal media plates, supplemented with antibiotics, IPTG and 3-AT as above, with and without 6.5 M l-arabinose. Plasmids from strains showing a growth advantage in the presence of arabinose were isolated and retransformed into the original selection strain and reassessed for IPTG-dependent inhibition of growth, and arabinose growth rescue. SICLOPPS plasmids from colonies demonstrating the expected phenotypes were assessed for their HIF-1 specificity by transformation into two identical RTHS, except for the replacement of HIF-1 with unrelated proteins (ATIC, a homodimeric enzyme used in purine biosynthesis, and P6/UEV, a heterodimeric interaction required for the budding of HIV from infected cells).24,25 Plasmids that caused a growth-advantage in the ATIC or P6/UEV RTHS were discarded for being nonspecific. The activity of the cyclic peptides encoded by the remaining SICLOPPS plasmids was ranked by retransforming into the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identity of the variable insert regions encoding the active cyclic peptides was revealed by DNA sequencing. Peptide Synthesis Cyclic peptides were synthesized and characterized as detailed in the Supporting Information. HIF Luciferase Reporter Assays Endogenous HIF-1 luciferase reporter assays were conducted as previously reported in U2OS-HRE-luc15 and MCF-7 cells.46 For plasmid-expressed HIF- luciferase reporter assays, MCF-7 and U2OS cells were transiently transfected with plasmids expressing HIF-1, HIF-2, or a blank control (pcDNA3.1-HIF-1, pcDNA3.1-HIF-2, or pcDNA3.1), a renilla-encoding control (phRL-TK), and a HIF-dependent firefly luciferase reporter construct (pGL2-TK-HRE), using Transfast (Promega) according to the manufacturers instructions. After 24 h, cells were recovered and plated (4000 cells/well) in 96-well plates (Perkin-Elmer) and incubated for 5 h before either hypoxic or aerobic incubation in presence or absence of cyclic peptide inhibitors. Firefly and renilla activities were determined using Dual-Glo Reagent (Promega) according to the manufacturers instructions. The luciferase signal was normalized using the corresponding renilla values. Recombinant Production of HIF-1 and HIF-1 HIF-1, HIF-2, HIF-1, bHLH, PAS-A, PAS-B, and PAS-B were expressed in (BL21.DE3) as detailed in the Supporting Information. In Vitro Assays Pull downs, ELISA, fluorescent binding assays, and ITC were conducted as detailed in the Assisting Info. Dosing Cells with Inhibitors Cells were treated with the stated concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia for 4 h, followed by incubation inside a hypoxic environment. All manipulation of cell pellets (e.g., lysis, mRNA, and protein extraction) was carried out inside a hypoxic environment. Duolink Proximity Ligation Assay Duolink proximity ligation assay was carried out using the in situ PLA Kit (O-Link Bioscience, Uppsala, Sweden) according to the manufacturers instructions. The antibodies used were rabbit monoclonal anti-HIF-1 (NB100-449, Novus Biologicals) and mouse monoclonal anti-HIF-1 (H00000405-B01P, Abnova). Cells were dosed with inhibitors as above and incubated inside a hypoxic environment for 4 h, after which they were fixed with.