hif-1α Search Results


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  • 94
    Abcam hif 1α
    Neutrophils and endothelial cells express <t>HIF-1α</t> within the ILD lung. Paraffin-embedded ILD lung biopsies were cut and stained for immunohistochemical evidence of hypoxia and neutrophil infiltration. (A) Slides stained for HIF-1α displayed positive brown staining within microvascular endothelial cells and polymorphonuclear cells, whilst (B) secondary antibody controls did not display positive staining. To verify whether neutrophils were present in the ILD lung, additional stains were performed for (C) MPO and (D) NE, both of which displayed positive brown stains within blood vessels. Abbreviations: HIF-1α, hypoxia-inducible factor 1α; MPO, myeloperoxidase; NE, neutrophil elastase.
    Hif 1α, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson hif 1α
    IMiD-induced FAM83F degradation occurs via the proteasome and is dependent on cereblon. (A) DLD-1 and HCT116 cell extracts treated with 10 μM IMiD compounds, 1 μM MLN4924, 5 μM Bortezomib or a combination thereof as indicated for 24 h were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. The accumulation of <t>HIF-1α</t> and ubiquitylated proteins following MLN4924 and Bortezomib treatments respectively were used as positive controls for successful compound treatments. (B) DLD-1 wild-type, DLD-1 CRBN -/- , DLD-1 CRBN -/- rescued with FLAG-CRBN and DLD-1 CRBN -/- rescued with FLAG-CRBN V388I cell extracts treated with IMiD compounds (10 μM 24 h), were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. (C) Densitometry of FAM83F protein abundance upon treatment with IMiD compounds (10 μM 24 h) from (B). FAM83F protein abundance was normalised to GAPDH protein abundance and represented as fold change compared to untreated cells. Data representative of two biological replicates with bar graph representing mean ± standard error.
    Hif 1α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology hif 1α
    Diagram of the proposed mechanism by which sotetsuflavone inhibits non-small-cell lung cancer A549 cell invasion and metastasis. Sotetsuflavone inhibited the invasion and metastasis of A549 cells by inhibiting EMT and angiogenesis. The anti-transfer effect of sotetsuflavone was mainly through downregulation of <t>HIF-1α</t> by the inhibition of PI3K/AKT and TNF-α/NF-κB signaling pathway in A549 cells, adjusting the whole process
    Hif 1α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc hif 1α
    Ubquitination of <t>HIF-1α</t> by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. ( n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. ( n = 6). **: P
    Hif 1α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Novus Biologicals hif 1 alpha antibody
    Ubquitination of <t>HIF-1α</t> by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. ( n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. ( n = 6). **: P
    Hif 1 Alpha Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 98/100, based on 1078 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals hif 1 alpha antibody h1alpha67
    Ubquitination of <t>HIF-1α</t> by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. ( n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. ( n = 6). **: P
    Hif 1 Alpha Antibody H1alpha67, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hif 1α
    Wheatgrass inhibits the hypoxia-mediated EMT process by inhibiting the activation of <t>HIF-1α</t> in airway epithelial cells. (A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h, and nuclear protein fractions were analyzed by immunoblotting with an anti-HIF-1α antibody. (B and C) Cells were transfected with either si-HIF-1α, si-negative control (NC), wheatgrass (150 µg/mL), or hypoxia only as indicated. E-cadherin expression levels (green) were analyzed by immunofluorescence analysis. (D and E) HIF-1α expression levels (red) were visualized by immunofluorescence analysis ( * P
    Hif 1α, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical hif 1α
    Smad3 association with the −335 HRE of the COL1A2 promoter is modulated by <t>HIF-1α.</t> For each DAPA, nuclear lysates from cells treated with 1 ng/ml TGF-β or vehicle for 6 h were used, and a representative of 3 experiments is shown.
    Hif 1α, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems human mouse rat hif 1 alpha antibody
    Smad3 association with the −335 HRE of the COL1A2 promoter is modulated by <t>HIF-1α.</t> For each DAPA, nuclear lysates from cells treated with 1 ng/ml TGF-β or vehicle for 6 h were used, and a representative of 3 experiments is shown.
    Human Mouse Rat Hif 1 Alpha Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson mouse anti hif 1α
    Effect of <t>HIF-1α</t> silencing on the hypoxia-induced protection against the etoposide-induced apoptosis. A , cells were transfected with 5, 20 or 50 nM HIF-1α siRNA, 50 nM non-targeting siRNA or with the transfection reagent alone (DMF) for 24 hours. Cells were then incubated under normoxia or hypoxia for 6 hours and total cell extracts were analyzed by western blot for HIF-1α protein level. A, B, C , cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. B , after the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction. C , caspase 3 activity was assayed. Results are expressed as means ± 1 SD (n = 3). *** p
    Mouse Anti Hif 1α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare anti hif 1α
    Hypoxia and <t>HIF-1α</t> downregulates the transcription of gene encoding for the PKA regulatory subunit RIIB. a Expression of the genes encoding for PKA regulatory and catalytic subunits in GH3 cells grown under hypoxic conditions (1% O 2 for 18 h) as determined by real-time RT-PCR. Data are
    Anti Hif 1α, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Neutrophils and endothelial cells express HIF-1α within the ILD lung. Paraffin-embedded ILD lung biopsies were cut and stained for immunohistochemical evidence of hypoxia and neutrophil infiltration. (A) Slides stained for HIF-1α displayed positive brown staining within microvascular endothelial cells and polymorphonuclear cells, whilst (B) secondary antibody controls did not display positive staining. To verify whether neutrophils were present in the ILD lung, additional stains were performed for (C) MPO and (D) NE, both of which displayed positive brown stains within blood vessels. Abbreviations: HIF-1α, hypoxia-inducible factor 1α; MPO, myeloperoxidase; NE, neutrophil elastase.

    Journal: bioRxiv

    Article Title: Identification of a novel HIF-1α-αMβ2 Integrin-NETosis axis in fibrotic interstitial lung disease

    doi: 10.1101/2020.01.03.894196

    Figure Lengend Snippet: Neutrophils and endothelial cells express HIF-1α within the ILD lung. Paraffin-embedded ILD lung biopsies were cut and stained for immunohistochemical evidence of hypoxia and neutrophil infiltration. (A) Slides stained for HIF-1α displayed positive brown staining within microvascular endothelial cells and polymorphonuclear cells, whilst (B) secondary antibody controls did not display positive staining. To verify whether neutrophils were present in the ILD lung, additional stains were performed for (C) MPO and (D) NE, both of which displayed positive brown stains within blood vessels. Abbreviations: HIF-1α, hypoxia-inducible factor 1α; MPO, myeloperoxidase; NE, neutrophil elastase.

    Article Snippet: HIF-1α (clone EP1215Y, Abcam, 1:600 dilution), MPO (polyclonal, Dako, 1:300 dilution) or NE (clone NP57, Dako, 1:100 dilution) was incubated in Epitope Retrieval Solution 2 for 20 minutes and stained using the 30,20,20 protocol.

    Techniques: Staining, Immunohistochemistry

    Hypoxia enhances NETosis but not hydrogen peroxide production. The effects of hypoxia upon neutrophil activation was first assessed. (A) The induction of hypoxia was verified by Western blot, probing for HIF-1α and HIF-2α. (B) NETosis was then evaluated by capture ELISA, which detects MPO-citrullinated histone H3 complexes. Data are presented as the mean and SEM from three independent experiments and analysed by two-way ANOVA with a Dunnett’s multiple comparison test. (C) Hydrogen peroxide generation was examined using Amplex ® UltraRed in absence and presence of 50nM PMA. Data are presented as the mean and SEM neutrophils isolated from 7 different donors and analysed by two-way ANOVA with a Dunnett’s multiple comparison test. *= p

    Journal: bioRxiv

    Article Title: Identification of a novel HIF-1α-αMβ2 Integrin-NETosis axis in fibrotic interstitial lung disease

    doi: 10.1101/2020.01.03.894196

    Figure Lengend Snippet: Hypoxia enhances NETosis but not hydrogen peroxide production. The effects of hypoxia upon neutrophil activation was first assessed. (A) The induction of hypoxia was verified by Western blot, probing for HIF-1α and HIF-2α. (B) NETosis was then evaluated by capture ELISA, which detects MPO-citrullinated histone H3 complexes. Data are presented as the mean and SEM from three independent experiments and analysed by two-way ANOVA with a Dunnett’s multiple comparison test. (C) Hydrogen peroxide generation was examined using Amplex ® UltraRed in absence and presence of 50nM PMA. Data are presented as the mean and SEM neutrophils isolated from 7 different donors and analysed by two-way ANOVA with a Dunnett’s multiple comparison test. *= p

    Article Snippet: HIF-1α (clone EP1215Y, Abcam, 1:600 dilution), MPO (polyclonal, Dako, 1:300 dilution) or NE (clone NP57, Dako, 1:100 dilution) was incubated in Epitope Retrieval Solution 2 for 20 minutes and stained using the 30,20,20 protocol.

    Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation

    Comparison of induction of HIF-1α and GLUT-1 between LoVo and HT29 cells/tumor tissues. ( A ) Micrographs of immunofluorescence staining for nuclei (blue) and HIF-1α/GLUT-1 (green) in LoVo (upper panel) and HT29 (lower panel) cells (magnification, 200×). ( B ) and ( C ) Quantification of HIF-1α and GLUT-1 staining using Live cell Imaging System. ( D ) Immunohistochemical staining for GLUT-1/HIF-1α (brown) in LoVo (upper panel) and HT29 (lower panel) xenograft tumor tissues (magnification, 200×), respectively. ( E ) Western blot analyses of HIF-1α and GLUT-1 protein expression in LoVo (right lane) and HT29 (left lane) xenograft tumor tissues. ( F ) Quantification of immunohistochemical staining. ( G ) Quantification of Western blot analyses. *** P

    Journal: Scientific Reports

    Article Title: Noninvasive evaluation of 18F-FDG/18F-FMISO-based Micro PET in monitoring hepatic metastasis of colorectal cancer

    doi: 10.1038/s41598-018-36238-x

    Figure Lengend Snippet: Comparison of induction of HIF-1α and GLUT-1 between LoVo and HT29 cells/tumor tissues. ( A ) Micrographs of immunofluorescence staining for nuclei (blue) and HIF-1α/GLUT-1 (green) in LoVo (upper panel) and HT29 (lower panel) cells (magnification, 200×). ( B ) and ( C ) Quantification of HIF-1α and GLUT-1 staining using Live cell Imaging System. ( D ) Immunohistochemical staining for GLUT-1/HIF-1α (brown) in LoVo (upper panel) and HT29 (lower panel) xenograft tumor tissues (magnification, 200×), respectively. ( E ) Western blot analyses of HIF-1α and GLUT-1 protein expression in LoVo (right lane) and HT29 (left lane) xenograft tumor tissues. ( F ) Quantification of immunohistochemical staining. ( G ) Quantification of Western blot analyses. *** P

    Article Snippet: Following 30 min of blocking with 10% normal goat serum, the cells were incubated with anti-HIF-1α (1:100; H1alpha67; Abcam, UK) or anti-GLUT-1 (1:100; ab40084; Abcam, UK) .

    Techniques: Immunofluorescence, Staining, Live Cell Imaging, Immunohistochemistry, Western Blot, Expressing

    Correlation analyses between various parameters. ( A ) and ( B ) correlation of 18F-FDG uptake with GLUT-1 ( A ) and HIF-1α ( B ) expression in vitro . ( C , D ) correlation of 18 F-FMISO uptake with GLUT-1 ( C ) and HIF-1α ( D ) expression in vitro . ( E ) and ( F ) correlation of in vivo values of 18 F-FMISO SUVmax with GLUT-1 ( E ) and HIF-1α ( F ) expression in tumor tissues.

    Journal: Scientific Reports

    Article Title: Noninvasive evaluation of 18F-FDG/18F-FMISO-based Micro PET in monitoring hepatic metastasis of colorectal cancer

    doi: 10.1038/s41598-018-36238-x

    Figure Lengend Snippet: Correlation analyses between various parameters. ( A ) and ( B ) correlation of 18F-FDG uptake with GLUT-1 ( A ) and HIF-1α ( B ) expression in vitro . ( C , D ) correlation of 18 F-FMISO uptake with GLUT-1 ( C ) and HIF-1α ( D ) expression in vitro . ( E ) and ( F ) correlation of in vivo values of 18 F-FMISO SUVmax with GLUT-1 ( E ) and HIF-1α ( F ) expression in tumor tissues.

    Article Snippet: Following 30 min of blocking with 10% normal goat serum, the cells were incubated with anti-HIF-1α (1:100; H1alpha67; Abcam, UK) or anti-GLUT-1 (1:100; ab40084; Abcam, UK) .

    Techniques: Expressing, In Vitro, In Vivo

    Regulation of myocardial infarction-associated transcript (MIAT) by ALKBH1 and HIF1α under ox-LDL treatment. a – d Miat mRNA relative expression and correlation with ALKBH1 level in leukocytes and aortic root tissue in WD-induced AS mice ( n = 4/group). e qRT-PCR assay of MIAT mRNA level with ox-LDL treatment in HUVECs. f , g Western blot assay of ox-LDL-induced ALKBH1 or HIF1α protein levels ( f ) and qRT-PCR assay of MIAT mRNA level ( g ) with siRNA targeting ALKBH1 or HIF1a pre-transfection in HUVECs. h qRT-PCR assay of MIAT mRNA level with ox-LDL treatment in THP1 cells. i , j Western blot assay of ox-LDL-induced ALKBH1 or HIF1α protein level ( i ) and qRT-PCR assay of MIAT mRNA level ( j ) with siRNA targeting ALKBH1 or HIF1a pre-transfection in THP1 cells. Data are mean ± SD ( n = 4/group) and were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test. * P

    Journal: Cell Death & Disease

    Article Title: Association of N6-methyladenine DNA with plaque progression in atherosclerosis via myocardial infarction-associated transcripts

    doi: 10.1038/s41419-019-2152-6

    Figure Lengend Snippet: Regulation of myocardial infarction-associated transcript (MIAT) by ALKBH1 and HIF1α under ox-LDL treatment. a – d Miat mRNA relative expression and correlation with ALKBH1 level in leukocytes and aortic root tissue in WD-induced AS mice ( n = 4/group). e qRT-PCR assay of MIAT mRNA level with ox-LDL treatment in HUVECs. f , g Western blot assay of ox-LDL-induced ALKBH1 or HIF1α protein levels ( f ) and qRT-PCR assay of MIAT mRNA level ( g ) with siRNA targeting ALKBH1 or HIF1a pre-transfection in HUVECs. h qRT-PCR assay of MIAT mRNA level with ox-LDL treatment in THP1 cells. i , j Western blot assay of ox-LDL-induced ALKBH1 or HIF1α protein level ( i ) and qRT-PCR assay of MIAT mRNA level ( j ) with siRNA targeting ALKBH1 or HIF1a pre-transfection in THP1 cells. Data are mean ± SD ( n = 4/group) and were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test. * P

    Article Snippet: The blots were incubated overnight with primary antibodies for N6AMT1 (Proteintech: 16211-1-AP), ALKBH1 (Abcam: ab195376), HIF1α (Abcam: ab1) and GAPDH (ab:8245) at 4 °C, then goat anti-rabbit or mouse IgG-HRP antibody at room temperature for 1 h. The positive bands were visualized by using Pierce ECL (ThermoFisher Scientific, USA) according to the manufacturer’s protocols.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Transfection

    Ox-LDL-induced m6A demethylation promotes HIF1α binding to the MIAT promoter and enhances its activity. a Immunofluorescence staining of subcellular localization of ALKBH1 and HIF1α in HUVECs after treatment with 50 μg/ml ox-LDL. Scale bar: 50 µm. DAPI was a nuclear marker. b Integrative genomics viewer plots showing the increasing m6A peaks (labeled ChIP1–3) in human MIAT gene (hg19) region with ALKBH1 knockdown by siRNA. Two HIF1α motifs on MIAT promoter are numbered. c – h Chromatin immunoprecipitation (ChIP) assay with m6A ( c – e ) or HIF1α ( f , g ) antibody used for immunoprecipitation on MIAT fragments in treated HUVECs; normal IgG was an IP control ( n = 4 per group). h Serial deletion constructs of MIAT-Luc with or without HIF1α deletion and pRL vectors were co-transfected into HUVECs. Relative promoter activities were measured by dual-luciferase reporter assay, normalized to Renilla activity ( n = 5 per group). i Elevated luciferase activity in ox-LDL-treated HUVECs prevented by pre-treatment with Si-RNA-ALKBH1 or HIF1α for MIAT plasmids with HIF1α motif ( n = 4 per group). Data are mean ± SD and were analyzed by one-way ANOVA, Tukey’s test for ( c – i ). * P

    Journal: Cell Death & Disease

    Article Title: Association of N6-methyladenine DNA with plaque progression in atherosclerosis via myocardial infarction-associated transcripts

    doi: 10.1038/s41419-019-2152-6

    Figure Lengend Snippet: Ox-LDL-induced m6A demethylation promotes HIF1α binding to the MIAT promoter and enhances its activity. a Immunofluorescence staining of subcellular localization of ALKBH1 and HIF1α in HUVECs after treatment with 50 μg/ml ox-LDL. Scale bar: 50 µm. DAPI was a nuclear marker. b Integrative genomics viewer plots showing the increasing m6A peaks (labeled ChIP1–3) in human MIAT gene (hg19) region with ALKBH1 knockdown by siRNA. Two HIF1α motifs on MIAT promoter are numbered. c – h Chromatin immunoprecipitation (ChIP) assay with m6A ( c – e ) or HIF1α ( f , g ) antibody used for immunoprecipitation on MIAT fragments in treated HUVECs; normal IgG was an IP control ( n = 4 per group). h Serial deletion constructs of MIAT-Luc with or without HIF1α deletion and pRL vectors were co-transfected into HUVECs. Relative promoter activities were measured by dual-luciferase reporter assay, normalized to Renilla activity ( n = 5 per group). i Elevated luciferase activity in ox-LDL-treated HUVECs prevented by pre-treatment with Si-RNA-ALKBH1 or HIF1α for MIAT plasmids with HIF1α motif ( n = 4 per group). Data are mean ± SD and were analyzed by one-way ANOVA, Tukey’s test for ( c – i ). * P

    Article Snippet: The blots were incubated overnight with primary antibodies for N6AMT1 (Proteintech: 16211-1-AP), ALKBH1 (Abcam: ab195376), HIF1α (Abcam: ab1) and GAPDH (ab:8245) at 4 °C, then goat anti-rabbit or mouse IgG-HRP antibody at room temperature for 1 h. The positive bands were visualized by using Pierce ECL (ThermoFisher Scientific, USA) according to the manufacturer’s protocols.

    Techniques: Binding Assay, Activity Assay, Immunofluorescence, Staining, Marker, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation, Construct, Transfection, Luciferase, Reporter Assay

    ROS was significantly increased in hypoxia-induced injury in rat primary neuron cells. ( A ) ROS level was detected by flow cytometry after 0, 6, 12, and 24 hours of hypoxic culture. ( B ) The mRNA expression of DUOX1 was detected by Q-PCR after 0, 6, 12, and 24 hours of hypoxic culture. ( C ) The protein expression of HIF-1α and DUOX1 was detected by western blot after 0, 6, 12, and 24 hours of hypoxic culture. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Vitamin D Attenuates Hypoxia-Induced Injury in Rat Primary Neuron Cells through Downregulation of the Dual Oxidase 1 (DUOX1) Gene

    doi: 10.12659/MSM.925350

    Figure Lengend Snippet: ROS was significantly increased in hypoxia-induced injury in rat primary neuron cells. ( A ) ROS level was detected by flow cytometry after 0, 6, 12, and 24 hours of hypoxic culture. ( B ) The mRNA expression of DUOX1 was detected by Q-PCR after 0, 6, 12, and 24 hours of hypoxic culture. ( C ) The protein expression of HIF-1α and DUOX1 was detected by western blot after 0, 6, 12, and 24 hours of hypoxic culture. * P

    Article Snippet: After blocking in 5% skim milk (BYL40422, BD Biosciences, Franklin Lakes, NJ, USA) for 1 hour at 25°C, the blots were probed with primary antibodies against HIF-1α (1: 400, Ab1, Abcam), DUOX1 (1: 1000, Orb539256, Biorbyt), VDR (1: 1000, Ab109234, Abcam), NF-κB (1: 2000, Ab16502, Abcam), cleaved caspase-3 (1: 1000, AF6311, Affinity), H3 (1: 1000, Ab1791, Abcam), and GAPDH (1: 2000, #5174, Cell Signaling Technology) overnight at 4°C with gentle shaking.

    Techniques: Flow Cytometry, Expressing, Polymerase Chain Reaction, Western Blot

    mRNA expression of HIF-1α, VEGF, and eNO S gene in granulocytes of MPN patients. (A) HIF-1 α gene expression was significantly reduced in PV heterozygous (htz), homozygous (hom), and PMF without JAK2 V617F mutation (NM) patients, while (B) VEGF gene expression was reduced in PV homo, ET, and PMF NM patients compared to controls (c). (C) Increased eNOS gene expression in MPN patients is presented as direct ratio with controls using SYBR Green. Values are mean SEM ( n = 5–10 patients per individual JAK2 mutant allele burden: hom, htz, and NM). * P

    Journal: Molecular carcinogenesis

    Article Title: Angiogenic Factors Are Increased in Circulating Granulocytes and CD34+ Cells of Myeloproliferative Neoplasms

    doi: 10.1002/mc.22517

    Figure Lengend Snippet: mRNA expression of HIF-1α, VEGF, and eNO S gene in granulocytes of MPN patients. (A) HIF-1 α gene expression was significantly reduced in PV heterozygous (htz), homozygous (hom), and PMF without JAK2 V617F mutation (NM) patients, while (B) VEGF gene expression was reduced in PV homo, ET, and PMF NM patients compared to controls (c). (C) Increased eNOS gene expression in MPN patients is presented as direct ratio with controls using SYBR Green. Values are mean SEM ( n = 5–10 patients per individual JAK2 mutant allele burden: hom, htz, and NM). * P

    Article Snippet: The next step was incubation with anti-HIF-1α antibody (Abcam) anti-VEGF antibody (BD Pharmingen, Cambridge, UK) and anti-eNOS antibody (Santa Cruz Biotechnology) in a humidity chamber over night at RT.

    Techniques: Expressing, Peptide Mass Fingerprinting, Mutagenesis, SYBR Green Assay

    Immunocytochemical analysis of angiogenic factors positive cells in granulocytes of MPN patients according to JAK2 V617F mutant allele burden. Quantitative analysis of (A) HIF-1α, (B) VEGF, and (C) eNOS percentage of positive cells in granulocytes revealed its significant increase in MPN patients heterozygous (htz), homozygous (hom), or no JAK2 V617F mutation (NM) versus controls (c), n = 5–6 patients per individual JAK2 mutant allele burden and controls. Values are mean ± SEM. ** P

    Journal: Molecular carcinogenesis

    Article Title: Angiogenic Factors Are Increased in Circulating Granulocytes and CD34+ Cells of Myeloproliferative Neoplasms

    doi: 10.1002/mc.22517

    Figure Lengend Snippet: Immunocytochemical analysis of angiogenic factors positive cells in granulocytes of MPN patients according to JAK2 V617F mutant allele burden. Quantitative analysis of (A) HIF-1α, (B) VEGF, and (C) eNOS percentage of positive cells in granulocytes revealed its significant increase in MPN patients heterozygous (htz), homozygous (hom), or no JAK2 V617F mutation (NM) versus controls (c), n = 5–6 patients per individual JAK2 mutant allele burden and controls. Values are mean ± SEM. ** P

    Article Snippet: The next step was incubation with anti-HIF-1α antibody (Abcam) anti-VEGF antibody (BD Pharmingen, Cambridge, UK) and anti-eNOS antibody (Santa Cruz Biotechnology) in a humidity chamber over night at RT.

    Techniques: Mutagenesis

    Immunocytochemical analysis of angiogenic factors positive cells in granulocytes of MPN patients after at least 6 months therapy with hydroxyurea (HU). A significant decrease in percentage of (A) HIF-1α, (B) VEGF, and (C) eNOS positive cells in ET, PV, and PMF ( n = 3× 6 patients before and after therapy with HU). Values are mean ± SEM. *** P

    Journal: Molecular carcinogenesis

    Article Title: Angiogenic Factors Are Increased in Circulating Granulocytes and CD34+ Cells of Myeloproliferative Neoplasms

    doi: 10.1002/mc.22517

    Figure Lengend Snippet: Immunocytochemical analysis of angiogenic factors positive cells in granulocytes of MPN patients after at least 6 months therapy with hydroxyurea (HU). A significant decrease in percentage of (A) HIF-1α, (B) VEGF, and (C) eNOS positive cells in ET, PV, and PMF ( n = 3× 6 patients before and after therapy with HU). Values are mean ± SEM. *** P

    Article Snippet: The next step was incubation with anti-HIF-1α antibody (Abcam) anti-VEGF antibody (BD Pharmingen, Cambridge, UK) and anti-eNOS antibody (Santa Cruz Biotechnology) in a humidity chamber over night at RT.

    Techniques: Peptide Mass Fingerprinting

    Immunohistochemical analysis of angiogenic factors positive cells in the bone marrow of MPN patients. (A) Increased percentage of cytoplasmatic HIF-1α positive cells is apparent in PV htz and PMF no JAK2 V617F mutation (NM) patients presented in adjacent immunohistochemical graphs. (B) It has been detected a significant increase in percentage of VEGF positive cells in ET htz and decrease in PV htz patients, while (C) eNOS positive cells are increased in PMF htz patients and reduced in ET NM patients versus controls (c). Arrows indicate positive brown cells. Corresponding controls are at the bottom left corner of immunohistochemical graphs. Bar 50 μm. Values are mean ± SEM ( n = 5–6 patients per individual JAK2 mutant allele burden). * P

    Journal: Molecular carcinogenesis

    Article Title: Angiogenic Factors Are Increased in Circulating Granulocytes and CD34+ Cells of Myeloproliferative Neoplasms

    doi: 10.1002/mc.22517

    Figure Lengend Snippet: Immunohistochemical analysis of angiogenic factors positive cells in the bone marrow of MPN patients. (A) Increased percentage of cytoplasmatic HIF-1α positive cells is apparent in PV htz and PMF no JAK2 V617F mutation (NM) patients presented in adjacent immunohistochemical graphs. (B) It has been detected a significant increase in percentage of VEGF positive cells in ET htz and decrease in PV htz patients, while (C) eNOS positive cells are increased in PMF htz patients and reduced in ET NM patients versus controls (c). Arrows indicate positive brown cells. Corresponding controls are at the bottom left corner of immunohistochemical graphs. Bar 50 μm. Values are mean ± SEM ( n = 5–6 patients per individual JAK2 mutant allele burden). * P

    Article Snippet: The next step was incubation with anti-HIF-1α antibody (Abcam) anti-VEGF antibody (BD Pharmingen, Cambridge, UK) and anti-eNOS antibody (Santa Cruz Biotechnology) in a humidity chamber over night at RT.

    Techniques: Immunohistochemistry, Peptide Mass Fingerprinting, Mutagenesis

    IMiD-induced FAM83F degradation occurs via the proteasome and is dependent on cereblon. (A) DLD-1 and HCT116 cell extracts treated with 10 μM IMiD compounds, 1 μM MLN4924, 5 μM Bortezomib or a combination thereof as indicated for 24 h were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. The accumulation of HIF-1α and ubiquitylated proteins following MLN4924 and Bortezomib treatments respectively were used as positive controls for successful compound treatments. (B) DLD-1 wild-type, DLD-1 CRBN -/- , DLD-1 CRBN -/- rescued with FLAG-CRBN and DLD-1 CRBN -/- rescued with FLAG-CRBN V388I cell extracts treated with IMiD compounds (10 μM 24 h), were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. (C) Densitometry of FAM83F protein abundance upon treatment with IMiD compounds (10 μM 24 h) from (B). FAM83F protein abundance was normalised to GAPDH protein abundance and represented as fold change compared to untreated cells. Data representative of two biological replicates with bar graph representing mean ± standard error.

    Journal: bioRxiv

    Article Title: IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

    doi: 10.1101/2020.05.25.114660

    Figure Lengend Snippet: IMiD-induced FAM83F degradation occurs via the proteasome and is dependent on cereblon. (A) DLD-1 and HCT116 cell extracts treated with 10 μM IMiD compounds, 1 μM MLN4924, 5 μM Bortezomib or a combination thereof as indicated for 24 h were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. The accumulation of HIF-1α and ubiquitylated proteins following MLN4924 and Bortezomib treatments respectively were used as positive controls for successful compound treatments. (B) DLD-1 wild-type, DLD-1 CRBN -/- , DLD-1 CRBN -/- rescued with FLAG-CRBN and DLD-1 CRBN -/- rescued with FLAG-CRBN V388I cell extracts treated with IMiD compounds (10 μM 24 h), were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies. (C) Densitometry of FAM83F protein abundance upon treatment with IMiD compounds (10 μM 24 h) from (B). FAM83F protein abundance was normalised to GAPDH protein abundance and represented as fold change compared to untreated cells. Data representative of two biological replicates with bar graph representing mean ± standard error.

    Article Snippet: Additional antibodies used were FAM83G (ab121750, Abcam), Alpha-tubulin (MA1-80189, Thermo Fisher Scientific), Ubiquitin (BML-PW8810, Enzo), HIF-1α (6109590, BD biosciences) and ZFP91 (A303-245A, Bethyl laboratories).

    Techniques: SDS Page, Western Blot

    Increased HIF-1α expression in Commd1 −/− embryos and regulation of HIF-1-mediated transcription by COMMD1. (A) Immunoblot analysis of HIF-1α (arrow) and tubulin in 9.5-dpc embryos and in NIH 3T3 cells either left untreated

    Journal:

    Article Title: Increased Activity of Hypoxia-Inducible Factor 1 Is Associated with Early Embryonic Lethality in Commd1 Null Mice ▿

    doi: 10.1128/MCB.01932-06

    Figure Lengend Snippet: Increased HIF-1α expression in Commd1 −/− embryos and regulation of HIF-1-mediated transcription by COMMD1. (A) Immunoblot analysis of HIF-1α (arrow) and tubulin in 9.5-dpc embryos and in NIH 3T3 cells either left untreated

    Article Snippet: HIF-1α protein expression in HEK 293T cells was detected with anti-HIF-1α (clone 54; BD Bioscience, Alphen aan den Rijn, The Netherlands).

    Techniques: Expressing

    Increased HIF-1α protein stability in COMMD1 knockdown cells and association of COMMD1 with HIF-1α. (A) The stability of HIF-1α protein was determined in HEK 293T COMMD1 knockdown (KD) and control (EV) cells by immunoblot analysis.

    Journal:

    Article Title: Increased Activity of Hypoxia-Inducible Factor 1 Is Associated with Early Embryonic Lethality in Commd1 Null Mice ▿

    doi: 10.1128/MCB.01932-06

    Figure Lengend Snippet: Increased HIF-1α protein stability in COMMD1 knockdown cells and association of COMMD1 with HIF-1α. (A) The stability of HIF-1α protein was determined in HEK 293T COMMD1 knockdown (KD) and control (EV) cells by immunoblot analysis.

    Article Snippet: HIF-1α protein expression in HEK 293T cells was detected with anti-HIF-1α (clone 54; BD Bioscience, Alphen aan den Rijn, The Netherlands).

    Techniques:

    Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Impaired Fetoplacental Angiogenesis in Growth-Restricted Fetuses With Abnormal Umbilical Artery Doppler Velocimetry Is Mediated by Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)

    doi: 10.1210/jc.2014-2385

    Figure Lengend Snippet: Heterodimer formation and transcriptional regulation after ARNT knockdown. A, Representative IP-IB demonstrates that after ARNT knockdown, IP with either HIF1α or ARNT results in decreased heterodimer formation. B, This results in decreased binding

    Article Snippet: Proteins were transferred to a polyvinylidene difluoride membrane and probed with antibodies against ARNT (BD Biosciences) or HIF1α (BD Biosciences).

    Techniques: Binding Assay

    VCE-004.8 mediates HIF-1α stabilization. a NIH3T3-EPO-Luc cells were stimulated with VCE-004.8 at the doses indicated for 6 h and assayed for luciferase activity. b Cells were pre-treated with VCE-004.8 for 1 h and then washed or not with PBS and incubated in complete medium for 6 h. Fold induction relative to untreated cells is shown. Data represent the mean ± SD ( n = 5). *** p

    Journal: Journal of Neuroinflammation

    Article Title: Hypoxia mimetic activity of VCE-004.8, a cannabidiol quinone derivative: implications for multiple sclerosis therapy

    doi: 10.1186/s12974-018-1103-y

    Figure Lengend Snippet: VCE-004.8 mediates HIF-1α stabilization. a NIH3T3-EPO-Luc cells were stimulated with VCE-004.8 at the doses indicated for 6 h and assayed for luciferase activity. b Cells were pre-treated with VCE-004.8 for 1 h and then washed or not with PBS and incubated in complete medium for 6 h. Fold induction relative to untreated cells is shown. Data represent the mean ± SD ( n = 5). *** p

    Article Snippet: Immunodetection of specific proteins was carried out by incubation with primary antibody against HIF-1α (1:1000; BD Biosciences, #610959 San Jose, CA, USA), HIF-2α (1:1000; Novus Biologicals, Littleton, USA), PHD1 (1:1000; Abcam, Cambridge, UK), PHD2 (1:1000; Abcam), PHD3 (1:1000; Abcam), OH-HIF-1α (1:1000; Cell Signaling, Danvers, MA, USA), PPARγ (1:1000), β-actin (1:10.000; Sigma), and arginase 1 (N20) (1:500; Santa Cruz, Dallas, TX, USA) overnight at 4 °C.

    Techniques: Luciferase, Activity Assay, Incubation

    Effect of VCE-004.8 on PDH activity and HIF-1α hydroxylation and stabilization. a MO3.13 cells were treated with VCE-004.8 for 6 h in the presence of MG132 and the levels of hydroxylated HIF-1α and HIF-1α were determined by immunoblot ( n = 3). b HEK-293T cells were transfected with HA-PHD1, HA-PHD2, or HA-PHD3 as indicated. After 24 h of transfection, cells were treated as follows: P1, non-transfected cells; P2, cells were transfected with PHDs and immunoprecipitated with IgG-HA; P3, cells were transfected with PHDs and immunoprecipitated with αHA; P4, cells transfected with PHDs, stimulated with VCE-004.8 (2.5 μM) and immunoprecipitated with αHA; P5, cells transfected with PHDs, stimulated with CBD (2.5 μM) and immunoprecipitated with αHA; P6, cells transfected with PHDs, stimulated with DMOG (1 mM) and immunoprecipitated with αHA. HIF prolyl hydroxylase activity was measured using GST-HIF-1α protein, and the levels of hydroxylated HIF-1α, HIF-1α, and PHDs were analyzed by immunoblot ( n = 3)

    Journal: Journal of Neuroinflammation

    Article Title: Hypoxia mimetic activity of VCE-004.8, a cannabidiol quinone derivative: implications for multiple sclerosis therapy

    doi: 10.1186/s12974-018-1103-y

    Figure Lengend Snippet: Effect of VCE-004.8 on PDH activity and HIF-1α hydroxylation and stabilization. a MO3.13 cells were treated with VCE-004.8 for 6 h in the presence of MG132 and the levels of hydroxylated HIF-1α and HIF-1α were determined by immunoblot ( n = 3). b HEK-293T cells were transfected with HA-PHD1, HA-PHD2, or HA-PHD3 as indicated. After 24 h of transfection, cells were treated as follows: P1, non-transfected cells; P2, cells were transfected with PHDs and immunoprecipitated with IgG-HA; P3, cells were transfected with PHDs and immunoprecipitated with αHA; P4, cells transfected with PHDs, stimulated with VCE-004.8 (2.5 μM) and immunoprecipitated with αHA; P5, cells transfected with PHDs, stimulated with CBD (2.5 μM) and immunoprecipitated with αHA; P6, cells transfected with PHDs, stimulated with DMOG (1 mM) and immunoprecipitated with αHA. HIF prolyl hydroxylase activity was measured using GST-HIF-1α protein, and the levels of hydroxylated HIF-1α, HIF-1α, and PHDs were analyzed by immunoblot ( n = 3)

    Article Snippet: Immunodetection of specific proteins was carried out by incubation with primary antibody against HIF-1α (1:1000; BD Biosciences, #610959 San Jose, CA, USA), HIF-2α (1:1000; Novus Biologicals, Littleton, USA), PHD1 (1:1000; Abcam, Cambridge, UK), PHD2 (1:1000; Abcam), PHD3 (1:1000; Abcam), OH-HIF-1α (1:1000; Cell Signaling, Danvers, MA, USA), PPARγ (1:1000), β-actin (1:10.000; Sigma), and arginase 1 (N20) (1:500; Santa Cruz, Dallas, TX, USA) overnight at 4 °C.

    Techniques: Activity Assay, Transfection, Immunoprecipitation

    Functional consequences of VCE-004.8 on HIF-1α stabilization. a PrimeKit co-cultures were seeded on day 0 and the indicated concentration of rhVEGFA or VCE-004.8 was added on day 2. Tube formation was analyzed, and the results were plotted using the Incucyte FLR software in terms of network length on day 7 ± SD ( n = 3). ** p

    Journal: Journal of Neuroinflammation

    Article Title: Hypoxia mimetic activity of VCE-004.8, a cannabidiol quinone derivative: implications for multiple sclerosis therapy

    doi: 10.1186/s12974-018-1103-y

    Figure Lengend Snippet: Functional consequences of VCE-004.8 on HIF-1α stabilization. a PrimeKit co-cultures were seeded on day 0 and the indicated concentration of rhVEGFA or VCE-004.8 was added on day 2. Tube formation was analyzed, and the results were plotted using the Incucyte FLR software in terms of network length on day 7 ± SD ( n = 3). ** p

    Article Snippet: Immunodetection of specific proteins was carried out by incubation with primary antibody against HIF-1α (1:1000; BD Biosciences, #610959 San Jose, CA, USA), HIF-2α (1:1000; Novus Biologicals, Littleton, USA), PHD1 (1:1000; Abcam, Cambridge, UK), PHD2 (1:1000; Abcam), PHD3 (1:1000; Abcam), OH-HIF-1α (1:1000; Cell Signaling, Danvers, MA, USA), PPARγ (1:1000), β-actin (1:10.000; Sigma), and arginase 1 (N20) (1:500; Santa Cruz, Dallas, TX, USA) overnight at 4 °C.

    Techniques: Functional Assay, Concentration Assay, Software

    Ror2 is regulated by HIF-1α and HIF-2α expression. A , knockdown of either HIF-1α or HIF-2α subunits suppresses Ror2 expression. Cells constitutively expressing both HIF-1α and HIF-2α were subjected to lentiviral shRNA of each subunit and sorted by GFP expression. Three populations sorted for increasing GFP expression were assayed for each knockdown and compared with a nonspecific sequence ( shNS ). When HIF-2α levels were suppressed, HIF-1α levels remain constant, whereas Ror2 was down-regulated. Similarly, when HIF-1α levels were highly suppressed and HIF-2α was stable, Ror2 was also down-regulated. B , HIF-1α and HIF-2α knockdown each lead to a loss of ROR2 expression. qRT-PCR analysis demonstrated effective shRNA-mediated suppression of HIF -1α (**, p = 0.0074; *, p = 0.0165) or HIF-2α (**, p = 0.0002; *, p = 0.0277). Ror2 expression is regulated by both HIF factors at the transcriptional level (**, p = 0.0002 HIF-1α, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

    doi: 10.1074/jbc.M109.073924

    Figure Lengend Snippet: Ror2 is regulated by HIF-1α and HIF-2α expression. A , knockdown of either HIF-1α or HIF-2α subunits suppresses Ror2 expression. Cells constitutively expressing both HIF-1α and HIF-2α were subjected to lentiviral shRNA of each subunit and sorted by GFP expression. Three populations sorted for increasing GFP expression were assayed for each knockdown and compared with a nonspecific sequence ( shNS ). When HIF-2α levels were suppressed, HIF-1α levels remain constant, whereas Ror2 was down-regulated. Similarly, when HIF-1α levels were highly suppressed and HIF-2α was stable, Ror2 was also down-regulated. B , HIF-1α and HIF-2α knockdown each lead to a loss of ROR2 expression. qRT-PCR analysis demonstrated effective shRNA-mediated suppression of HIF -1α (**, p = 0.0074; *, p = 0.0165) or HIF-2α (**, p = 0.0002; *, p = 0.0277). Ror2 expression is regulated by both HIF factors at the transcriptional level (**, p = 0.0002 HIF-1α, p

    Article Snippet: The HIF-1α antibody was obtained from BD Transductions (Franklin Lakes, NJ) and the Glut1 and Egln3 antibodies were obtained from Novus Biologicals (Littleton, CO).

    Techniques: Expressing, shRNA, Sequencing, Quantitative RT-PCR

    HIF-2α and ARNT interact with the ROR2 promoter. Cells lacking expression of VHL and overexpressing only HIF-2α (786-0) were subjected to chromatin immunoprecipitation with HIF-1α (used as a negative control), HIF-2α, and ARNT antibodies. Primers were designed targeting the 1-kb region of the Ror2 promoter that overlaps the Ror2 start site. Using primers targeted against the promoter region containing a known hypoxic response element of PHD3 , also known as EGLN3 , we show that HIF-2α and its dimerization partner, ARNT, interact with the PHD3 promoter as expected. HIF-1α interaction is not detected, as this cell line does not express HIF-1α. When the ROR2 promoter was examined, HIF-2α and ARNT were localized to ROR2 promoter regions B and C similarly to the control promoter. Interaction was not observed for flanking regions A and D .

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Ror2 as a Hypoxia-inducible Factor Target in von Hippel-Lindau-associated Renal Cell Carcinoma *

    doi: 10.1074/jbc.M109.073924

    Figure Lengend Snippet: HIF-2α and ARNT interact with the ROR2 promoter. Cells lacking expression of VHL and overexpressing only HIF-2α (786-0) were subjected to chromatin immunoprecipitation with HIF-1α (used as a negative control), HIF-2α, and ARNT antibodies. Primers were designed targeting the 1-kb region of the Ror2 promoter that overlaps the Ror2 start site. Using primers targeted against the promoter region containing a known hypoxic response element of PHD3 , also known as EGLN3 , we show that HIF-2α and its dimerization partner, ARNT, interact with the PHD3 promoter as expected. HIF-1α interaction is not detected, as this cell line does not express HIF-1α. When the ROR2 promoter was examined, HIF-2α and ARNT were localized to ROR2 promoter regions B and C similarly to the control promoter. Interaction was not observed for flanking regions A and D .

    Article Snippet: The HIF-1α antibody was obtained from BD Transductions (Franklin Lakes, NJ) and the Glut1 and Egln3 antibodies were obtained from Novus Biologicals (Littleton, CO).

    Techniques: Expressing, Chromatin Immunoprecipitation, Negative Control

    Diagram of the proposed mechanism by which sotetsuflavone inhibits non-small-cell lung cancer A549 cell invasion and metastasis. Sotetsuflavone inhibited the invasion and metastasis of A549 cells by inhibiting EMT and angiogenesis. The anti-transfer effect of sotetsuflavone was mainly through downregulation of HIF-1α by the inhibition of PI3K/AKT and TNF-α/NF-κB signaling pathway in A549 cells, adjusting the whole process

    Journal: Cell Death Discovery

    Article Title: Sotetsuflavone suppresses invasion and metastasis in non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling pathway

    doi: 10.1038/s41420-018-0026-9

    Figure Lengend Snippet: Diagram of the proposed mechanism by which sotetsuflavone inhibits non-small-cell lung cancer A549 cell invasion and metastasis. Sotetsuflavone inhibited the invasion and metastasis of A549 cells by inhibiting EMT and angiogenesis. The anti-transfer effect of sotetsuflavone was mainly through downregulation of HIF-1α by the inhibition of PI3K/AKT and TNF-α/NF-κB signaling pathway in A549 cells, adjusting the whole process

    Article Snippet: HIF-1α purchased from Santa Cruz Biotechnology (Shanghai, China).

    Techniques: Inhibition

    Sotetsuflavone inhibited HIF-1α expression. a Immunofluorescence assay detected the expression of HIF-1α. Scale bar 50 μm. b Western blotting detected HIF-1α expression. Using the same sample lysates as used for Fig. 2a , another gel (gel B) was run and probed with anti- HIF-1α. The GAPDH loading control is from gel A, and is the same as shown in Fig. 2a . The data in the histogram represent the mean ± SD from three independent experiments (** P

    Journal: Cell Death Discovery

    Article Title: Sotetsuflavone suppresses invasion and metastasis in non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling pathway

    doi: 10.1038/s41420-018-0026-9

    Figure Lengend Snippet: Sotetsuflavone inhibited HIF-1α expression. a Immunofluorescence assay detected the expression of HIF-1α. Scale bar 50 μm. b Western blotting detected HIF-1α expression. Using the same sample lysates as used for Fig. 2a , another gel (gel B) was run and probed with anti- HIF-1α. The GAPDH loading control is from gel A, and is the same as shown in Fig. 2a . The data in the histogram represent the mean ± SD from three independent experiments (** P

    Article Snippet: HIF-1α purchased from Santa Cruz Biotechnology (Shanghai, China).

    Techniques: Expressing, Immunofluorescence, Western Blot

    HIF-1α and NOS2 expression in lesion tissue. Three days after beginning of the stress protocol, a full-thickness excisional lesion (1 cm 2 ) was generated on the dorsal skin. Seven days after wounding, lesion was collected and proteins were resolved

    Journal: Experimental Biology and Medicine

    Article Title: Blockade of glucocorticoid receptors improves cutaneous wound healing in stressed mice

    doi: 10.1177/1535370215612940

    Figure Lengend Snippet: HIF-1α and NOS2 expression in lesion tissue. Three days after beginning of the stress protocol, a full-thickness excisional lesion (1 cm 2 ) was generated on the dorsal skin. Seven days after wounding, lesion was collected and proteins were resolved

    Article Snippet: Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) and probed with anti-HIF-1α, anti-inducible nitric oxide synthase (NOS2), and anti-β-actin antibodies (Santa Cruz Biotechnology) followed by biotin-conjugated antibody and streptavidin-conjugated horseradish peroxidase developed by chemiluminescence (Santa Cruz Biotechnology).

    Techniques: Expressing, Generated

    Double immunofluorescent detection of glucagon with ATF3, HIF1α, or FHL1 in the pancreatic islets of PC2 null or wild-type littermate mice. Pancreas sections of PC2 null ( d–f , j–l , and p – r ) and wild-type ( a–c , g–i , and m – o ) mice were immunostained with antiglucagon ( a , d , g , j , m , and p ), anti-ATF3 ( b and e ), anti-HIF1α ( h and k ), and anti-FHL1 ( n and q ) antibodies. c , f , i , l , o , and r are images of double staining of a and b , d and e , g and h , j and k , m and n , and p and q , respectively. (Bar = 50 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Contrasting patterns of expression of transcription factors in pancreatic ? and ? cells

    doi: 10.1073/pnas.1735286100

    Figure Lengend Snippet: Double immunofluorescent detection of glucagon with ATF3, HIF1α, or FHL1 in the pancreatic islets of PC2 null or wild-type littermate mice. Pancreas sections of PC2 null ( d–f , j–l , and p – r ) and wild-type ( a–c , g–i , and m – o ) mice were immunostained with antiglucagon ( a , d , g , j , m , and p ), anti-ATF3 ( b and e ), anti-HIF1α ( h and k ), and anti-FHL1 ( n and q ) antibodies. c , f , i , l , o , and r are images of double staining of a and b , d and e , g and h , j and k , m and n , and p and q , respectively. (Bar = 50 μm.)

    Article Snippet: Rabbit antiserum to ATF3, goat antisera to HIF1α, and FHL1 (Santa Cruz Biotechnology), as well as guinea pig antiglucagon (Linco Research Immunoassay, St. Charles, MO), were purchased commercially.

    Techniques: Mouse Assay, Double Staining

    The effect of gallic acid (GA) on HIF-1α and HIF-1β protein expression in OVCAR-3 cells and luciferase assay after transfection with VEGF luciferase reporter and HIF-1α plasmids. (A) GA decreases HIF-1α protein expression. Data represents mean ± SE from 4 independent experiment. (B) HIF-1α mediates the inhibitory effect by GA on VEGF transcriptional activation. Data represents means ± SE from 6 independent experiments. ** P

    Journal: Oncology Reports

    Article Title: Gallic acid, a phenolic compound, exerts anti-angiogenic effects via the PTEN/AKT/HIF-1α/VEGF signaling pathway in ovarian cancer cells

    doi: 10.3892/or.2015.4354

    Figure Lengend Snippet: The effect of gallic acid (GA) on HIF-1α and HIF-1β protein expression in OVCAR-3 cells and luciferase assay after transfection with VEGF luciferase reporter and HIF-1α plasmids. (A) GA decreases HIF-1α protein expression. Data represents mean ± SE from 4 independent experiment. (B) HIF-1α mediates the inhibitory effect by GA on VEGF transcriptional activation. Data represents means ± SE from 6 independent experiments. ** P

    Article Snippet: For immunodetection, antibodies against HIF-1α, HIF-1β, AKT, p-AKT, PTEN and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied, and signals were visualized with phycoerythrin-conjugated anti-mouse IgG secondary antibodies, SuperSignal West Pico Substrate, and X-ray film (Pierce).

    Techniques: Expressing, Luciferase, Transfection, Activation Assay

    The effect of gallic acid (GA) on p-AKT and AKT protein expression in OVCAR-3 cells and luciferase assay after transfection with mAKT plasmids and VEGF luciferase reporter or HIF-1α luciferase reporter. (A) GA decreases p-AKT protein expression in OVCAR-3 cells. Data represents mean ± SE from 5 independent experiment. (B) mAKT mediates the inhibitory effects of GA on VEGF expression in ovarian cancer cells. Data represents mean ± SE from 8 independent experiment. (C) mAKT mediates the inhibitory effects of GA on HIF-1α expression in ovarian cancer cells. Data represents mean ± SE from 4 independent experiments. * P

    Journal: Oncology Reports

    Article Title: Gallic acid, a phenolic compound, exerts anti-angiogenic effects via the PTEN/AKT/HIF-1α/VEGF signaling pathway in ovarian cancer cells

    doi: 10.3892/or.2015.4354

    Figure Lengend Snippet: The effect of gallic acid (GA) on p-AKT and AKT protein expression in OVCAR-3 cells and luciferase assay after transfection with mAKT plasmids and VEGF luciferase reporter or HIF-1α luciferase reporter. (A) GA decreases p-AKT protein expression in OVCAR-3 cells. Data represents mean ± SE from 5 independent experiment. (B) mAKT mediates the inhibitory effects of GA on VEGF expression in ovarian cancer cells. Data represents mean ± SE from 8 independent experiment. (C) mAKT mediates the inhibitory effects of GA on HIF-1α expression in ovarian cancer cells. Data represents mean ± SE from 4 independent experiments. * P

    Article Snippet: For immunodetection, antibodies against HIF-1α, HIF-1β, AKT, p-AKT, PTEN and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied, and signals were visualized with phycoerythrin-conjugated anti-mouse IgG secondary antibodies, SuperSignal West Pico Substrate, and X-ray film (Pierce).

    Techniques: Expressing, Luciferase, Transfection

    Immunohistochemical examination of HIF-1α in the ovaries from each group. HIF-1α immunohistochemical signals are brown and the hematoxylin counterstaining background are blue. Negative controls remained unstained as they lacked primary antibody. (A and B) HIF-1α immunohistochemical staining of ovaries from the control group. (C) Negative control of the control group. (D and E) HIF-1α immunohistochemical staining in ovaries from group A. (F) Negative control of group A. (G and H) HIF-1α immunohistochemistry in ovaries from group B. (I) Negative control of group B. (J and K) HIF-1α immunohistochemical staining in ovaries from group C. (L) Negative control of group C. Scale bar=100 µm. Coloured boxes on the left side panels indicate the area exhibited on the middle and right side panels. GC, granulosa cell, Oo; oocyte; control group, mice treated with corn oil; group A, mice treated with a low dose of DMC; group B, mice treated with a medium dose of DMC; group C, mice treated with a high dose of DMC; DMC, dimethyl carbonate.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Effects of dimethyl carbonate-induced autophagic activation on follicular development in the mouse ovary

    doi: 10.3892/etm.2017.5328

    Figure Lengend Snippet: Immunohistochemical examination of HIF-1α in the ovaries from each group. HIF-1α immunohistochemical signals are brown and the hematoxylin counterstaining background are blue. Negative controls remained unstained as they lacked primary antibody. (A and B) HIF-1α immunohistochemical staining of ovaries from the control group. (C) Negative control of the control group. (D and E) HIF-1α immunohistochemical staining in ovaries from group A. (F) Negative control of group A. (G and H) HIF-1α immunohistochemistry in ovaries from group B. (I) Negative control of group B. (J and K) HIF-1α immunohistochemical staining in ovaries from group C. (L) Negative control of group C. Scale bar=100 µm. Coloured boxes on the left side panels indicate the area exhibited on the middle and right side panels. GC, granulosa cell, Oo; oocyte; control group, mice treated with corn oil; group A, mice treated with a low dose of DMC; group B, mice treated with a medium dose of DMC; group C, mice treated with a high dose of DMC; DMC, dimethyl carbonate.

    Article Snippet: The primary antibodies used were anti-LC-3B antibody (1:500, cat. no. ab48394, Abcam, Cambridge, MA, USA) and anti-HIF-1α antibody (1:400, cat. no. sc-53546, Santa Cruz Biotechnology Inc., Dallas, TX, USA).

    Techniques: Immunohistochemistry, Staining, Negative Control, Mouse Assay

    Western blotting analysis of HIF-1α and BNIP3 expression in the ovaries from each group. (A) Western blotting indicating the expression of HIF-1α and BNIP3 in the different groups. (B) Western blotting indicating the expression of HIF-1α nuclear protein in the different groups. (C) Quantification of BNIP3 expression normalized to β-actin. (D) Quantification of total HIF-1α expression normalized to β-actin. (E) Quantification of HIF-1α nuclear expression normalized to β-actin. Data are expressed as the mean ± standard error of the mean. One-way analysis of variance was used to analyze the data. # P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Effects of dimethyl carbonate-induced autophagic activation on follicular development in the mouse ovary

    doi: 10.3892/etm.2017.5328

    Figure Lengend Snippet: Western blotting analysis of HIF-1α and BNIP3 expression in the ovaries from each group. (A) Western blotting indicating the expression of HIF-1α and BNIP3 in the different groups. (B) Western blotting indicating the expression of HIF-1α nuclear protein in the different groups. (C) Quantification of BNIP3 expression normalized to β-actin. (D) Quantification of total HIF-1α expression normalized to β-actin. (E) Quantification of HIF-1α nuclear expression normalized to β-actin. Data are expressed as the mean ± standard error of the mean. One-way analysis of variance was used to analyze the data. # P

    Article Snippet: The primary antibodies used were anti-LC-3B antibody (1:500, cat. no. ab48394, Abcam, Cambridge, MA, USA) and anti-HIF-1α antibody (1:400, cat. no. sc-53546, Santa Cruz Biotechnology Inc., Dallas, TX, USA).

    Techniques: Western Blot, Expressing

    Ubquitination of HIF-1α by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. ( n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. ( n = 6). **: P

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Ubquitination of HIF-1α by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. ( n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. ( n = 6). **: P

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Mutagenesis, Transfection, In Vivo, Multiple Displacement Amplification, Transduction, Luciferase, shRNA, Plasmid Preparation

    Low Parkin expression is correlated with increased HIF-1α levels and poor distant metastasis-free survival in human breast cancer. a , b Low Parkin expression was significantly correlated with increased HIF-1α levels in human breast cancer specimens analyzed by IHC staining. Upper panel in a : representative IHC staining images in TMA-RCINJ ( n = 200, from the RCINJ). Scale bar: 20 µm. Lower panel in a : summary of IHC staining results of human breast cancer specimens in TMA-RCINJ. b Summary of IHC staining results in two different cohorts of human breast cancer specimens in TMAs ( n = 120 in TMA-BR2082a; n = 220 in TMA-BR2281 from US Biomax). c , d Low Parkin expression was significantly associated with poor distant metastasis-free survival in human breast cancer. In c , low Parkin expression was significantly associated with poor distant metastasis-free survival in TMA-RCINJ. In d , the survival information and mRNA levels of Parkin were obtained from GEO databases. In c , d : Kaplan–Meier curves were drawn by GraphPad Prism software. Differences between the two survival curves were analyzed using the log-rank (Mantel–Cox) test. e A model depicting the suppression of cancer metastasis by Parkin-mediated HIF-1α ubiquitination and degradation

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Low Parkin expression is correlated with increased HIF-1α levels and poor distant metastasis-free survival in human breast cancer. a , b Low Parkin expression was significantly correlated with increased HIF-1α levels in human breast cancer specimens analyzed by IHC staining. Upper panel in a : representative IHC staining images in TMA-RCINJ ( n = 200, from the RCINJ). Scale bar: 20 µm. Lower panel in a : summary of IHC staining results of human breast cancer specimens in TMA-RCINJ. b Summary of IHC staining results in two different cohorts of human breast cancer specimens in TMAs ( n = 120 in TMA-BR2082a; n = 220 in TMA-BR2281 from US Biomax). c , d Low Parkin expression was significantly associated with poor distant metastasis-free survival in human breast cancer. In c , low Parkin expression was significantly associated with poor distant metastasis-free survival in TMA-RCINJ. In d , the survival information and mRNA levels of Parkin were obtained from GEO databases. In c , d : Kaplan–Meier curves were drawn by GraphPad Prism software. Differences between the two survival curves were analyzed using the log-rank (Mantel–Cox) test. e A model depicting the suppression of cancer metastasis by Parkin-mediated HIF-1α ubiquitination and degradation

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Expressing, Immunohistochemistry, Staining, Software

    Parkin inhibits migration and invasion of human breast cancer cells through negative regulation of HIF-1α. a , b Myc-Parkin expression inhibited the migration ( a ) and invasion ( b ) of different human breast cells as determined by transwell assays. Left panels of a , b : representative images of migrating ( a ) or invading ( b ) MDA-MB231 cells transduced with control (Con) or Myc-Parkin vectors. Scale bars: 200 μm. Right panels of a , b : quantification of average number of migrating or invading cells per field. c Knockdown of endogenous Parkin by shRNA vectors promoted the migration and invasion of different human breast cells. d Parkin −/− MEFs displayed enhanced abilities of migration and invasion compared with Parkin + / + MEFs. Left panel: representative images of migrating or invading cells. Scale bars: 500 μm; Right panel: quantification of average number of migrating or invading cells per field. e Knockdown of HIF-1α largely abolished the promoting effect of Parkin knockdown on migration (upper panel) and invasion (lower panel) of cells as measured by transwell assays. Cells with HIF-1α knockdown were further transduced with control or Parkin shRNA vectors for transwell assays. f Expression of K477R HA-rHIF-1α largely abolished the inhibitory effect of Myc-Parkin on cell migration (upper panel) and invasion (lower panel) in MCF7 and MDA-MB231 cells. Endogenous HIF-1α in cells was replaced with WT or K477R HA-rHIF-1α (shown in Fig. 5g ), and cells were then transduced with Myc-Parkin for transwell assays. g C431A, T173A, T240M and P294S mutations of Parkin compromised the inhibitory effects of Parkin on cell migration (left panel) and invasion (right panel). In a – g , the data present mean ± SD ( n = 6). #: P

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin inhibits migration and invasion of human breast cancer cells through negative regulation of HIF-1α. a , b Myc-Parkin expression inhibited the migration ( a ) and invasion ( b ) of different human breast cells as determined by transwell assays. Left panels of a , b : representative images of migrating ( a ) or invading ( b ) MDA-MB231 cells transduced with control (Con) or Myc-Parkin vectors. Scale bars: 200 μm. Right panels of a , b : quantification of average number of migrating or invading cells per field. c Knockdown of endogenous Parkin by shRNA vectors promoted the migration and invasion of different human breast cells. d Parkin −/− MEFs displayed enhanced abilities of migration and invasion compared with Parkin + / + MEFs. Left panel: representative images of migrating or invading cells. Scale bars: 500 μm; Right panel: quantification of average number of migrating or invading cells per field. e Knockdown of HIF-1α largely abolished the promoting effect of Parkin knockdown on migration (upper panel) and invasion (lower panel) of cells as measured by transwell assays. Cells with HIF-1α knockdown were further transduced with control or Parkin shRNA vectors for transwell assays. f Expression of K477R HA-rHIF-1α largely abolished the inhibitory effect of Myc-Parkin on cell migration (upper panel) and invasion (lower panel) in MCF7 and MDA-MB231 cells. Endogenous HIF-1α in cells was replaced with WT or K477R HA-rHIF-1α (shown in Fig. 5g ), and cells were then transduced with Myc-Parkin for transwell assays. g C431A, T173A, T240M and P294S mutations of Parkin compromised the inhibitory effects of Parkin on cell migration (left panel) and invasion (right panel). In a – g , the data present mean ± SD ( n = 6). #: P

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Migration, Expressing, Multiple Displacement Amplification, Transduction, shRNA

    Parkin inhibits lung metastasis of human breast cancer cells in vivo. a , b Myc-Parkin expression inhibited lung metastasis of breast cancer cells injected via the tail vein. MDA-MB231 cells with Myc-Parkin expression were transduced with lentiviral vectors expressing luciferase for tail vein injections. In a , left panel: representative bioluminescent images at indicated time points; right panel: normalized photon flux of lung metastases. In b , left panel: representative H E-stained lung sections from mice at 12 weeks after injections. Arrows indicate metastatic nodules. Scale bar: 200 μm. Middle panel: quantification of lung metastatic nodules. Right panel: IHC staining of Parkin and HIF-1α in lung metastases. Scale bar: 20 μm. c , d Knockdown of Parkin promoted lung metastasis of MCF7 cells injected via the tail vein. e HIF-1α knockdown largely abolished the promoting effects of Parkin knockdown on lung metastasis of MCF7 cells after tail vein injections. Two different Parkin shRNA vectors were used and similar results were observed. f K477R HA-rHIF-1α expression reduced the inhibitory effect of Myc-Parkin expression on lung metastasis of MDA-MB231 cells injected via the tail vein. Endogenous HIF-1α in cells was replaced with WT or K477R HA-rHIF-1α. g The HIF-1α inhibitor YC-1 largely abolished the promoting effect of Parkin knockdown on lung metastasis. Mice were treated with YC-1 (30 mg kg −1 per day; i.p.) for 5 days after the tail vein injection of MCF7 cells. h Myc-Parkin expression inhibited lung metastasis of MDA-MB231 cells implanted into mammary fat pads of mice. i K477R HA-rHIF-1α expression reduced the inhibitory effect of Myc-Parkin on lung metastasis of MDA-MB231 cells implanted into mammary fat pads. In h , i , primary tumors were surgically removed when they reached a volume of ~200 mm 3 . Left panels in h , i : representative H E-stained lung sections at 8 weeks after primary tumor removal. Right panels in h , i : quantification of lung metastatic nodules at 8 weeks after primary tumor removal. n = 6 mice per group in a – d , f ; n = 8 mice per group in e , g – i . The data present mean ± S.D. * P

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin inhibits lung metastasis of human breast cancer cells in vivo. a , b Myc-Parkin expression inhibited lung metastasis of breast cancer cells injected via the tail vein. MDA-MB231 cells with Myc-Parkin expression were transduced with lentiviral vectors expressing luciferase for tail vein injections. In a , left panel: representative bioluminescent images at indicated time points; right panel: normalized photon flux of lung metastases. In b , left panel: representative H E-stained lung sections from mice at 12 weeks after injections. Arrows indicate metastatic nodules. Scale bar: 200 μm. Middle panel: quantification of lung metastatic nodules. Right panel: IHC staining of Parkin and HIF-1α in lung metastases. Scale bar: 20 μm. c , d Knockdown of Parkin promoted lung metastasis of MCF7 cells injected via the tail vein. e HIF-1α knockdown largely abolished the promoting effects of Parkin knockdown on lung metastasis of MCF7 cells after tail vein injections. Two different Parkin shRNA vectors were used and similar results were observed. f K477R HA-rHIF-1α expression reduced the inhibitory effect of Myc-Parkin expression on lung metastasis of MDA-MB231 cells injected via the tail vein. Endogenous HIF-1α in cells was replaced with WT or K477R HA-rHIF-1α. g The HIF-1α inhibitor YC-1 largely abolished the promoting effect of Parkin knockdown on lung metastasis. Mice were treated with YC-1 (30 mg kg −1 per day; i.p.) for 5 days after the tail vein injection of MCF7 cells. h Myc-Parkin expression inhibited lung metastasis of MDA-MB231 cells implanted into mammary fat pads of mice. i K477R HA-rHIF-1α expression reduced the inhibitory effect of Myc-Parkin on lung metastasis of MDA-MB231 cells implanted into mammary fat pads. In h , i , primary tumors were surgically removed when they reached a volume of ~200 mm 3 . Left panels in h , i : representative H E-stained lung sections at 8 weeks after primary tumor removal. Right panels in h , i : quantification of lung metastatic nodules at 8 weeks after primary tumor removal. n = 6 mice per group in a – d , f ; n = 8 mice per group in e , g – i . The data present mean ± S.D. * P

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: In Vivo, Expressing, Injection, Multiple Displacement Amplification, Transduction, Luciferase, Staining, Mouse Assay, Immunohistochemistry, shRNA

    Parkin interacts with HIF-1α. a Parkin protein expression was significantly decreased in breast cancer specimens compared with non-tumor breast tissues as analyzed by IHC. Left panel: representative IHC staining images of Parkin in a human breast TMA. Scale bar: 20 μm. Right panel: summary of IHC staining of Parkin in breast cancer specimens ( n = 120) and non-tumor breast tissues ( n = 48) in a human breast TMA (TMA-BR2082a; US Biomax). b Parkin mRNA levels were significantly decreased in human breast cancers compared with matched adjacent non-tumor breast tissues ( n = 113). The data were obtained from TCGA. In a , b , P

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin interacts with HIF-1α. a Parkin protein expression was significantly decreased in breast cancer specimens compared with non-tumor breast tissues as analyzed by IHC. Left panel: representative IHC staining images of Parkin in a human breast TMA. Scale bar: 20 μm. Right panel: summary of IHC staining of Parkin in breast cancer specimens ( n = 120) and non-tumor breast tissues ( n = 48) in a human breast TMA (TMA-BR2082a; US Biomax). b Parkin mRNA levels were significantly decreased in human breast cancers compared with matched adjacent non-tumor breast tissues ( n = 113). The data were obtained from TCGA. In a , b , P

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Expressing, Immunohistochemistry, Staining

    Parkin negatively regulates HIF-1α protein levels in cells. a Myc-Parkin expression reduced levels of exogenous HA-HIF-1α protein in MCF7 and MDA-MB231 cells. Cells were transfected with the HA-HIF-1α vector together with varying amounts of Myc-Parkin or empty control (Con) vectors. b Ectopic Myc-Parkin expression reduced levels of endogenous HIF-1α protein in different human breast cells. c Knockdown of endogenous Parkin by 2 different shRNA vectors increased levels of endogenous HIF-1α protein in different human breast cells. d HIF-1α protein levels were higher in Parkin −/− MEFs compared with Parkin + / + MEFs. e Myc-Parkin expression downregulated HIF-1α protein levels in MCF7 cells under both normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions. f Knockdown of Parkin increased HIF-1α protein levels in MCF7 cells under both normoxic and hypoxic conditions. g Higher HIF-1α protein levels in Parkin − / − MEFs than Parkin + / + MEFs under both normoxic and hypoxic conditions. h Myc-Parkin expression reduced the levels of endogenous HIF-1α protein in both VHL-deficient RCC4 cells and VHL-proficient RCC4/VHL cells. i Knockdown of Parkin increased HIF-1α protein levels in both RCC4 and RCC4/VHL cells. j Knockdown of Parkin in MCF7 cells with VHL knockdown further increased HIF-1α protein levels

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin negatively regulates HIF-1α protein levels in cells. a Myc-Parkin expression reduced levels of exogenous HA-HIF-1α protein in MCF7 and MDA-MB231 cells. Cells were transfected with the HA-HIF-1α vector together with varying amounts of Myc-Parkin or empty control (Con) vectors. b Ectopic Myc-Parkin expression reduced levels of endogenous HIF-1α protein in different human breast cells. c Knockdown of endogenous Parkin by 2 different shRNA vectors increased levels of endogenous HIF-1α protein in different human breast cells. d HIF-1α protein levels were higher in Parkin −/− MEFs compared with Parkin + / + MEFs. e Myc-Parkin expression downregulated HIF-1α protein levels in MCF7 cells under both normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions. f Knockdown of Parkin increased HIF-1α protein levels in MCF7 cells under both normoxic and hypoxic conditions. g Higher HIF-1α protein levels in Parkin − / − MEFs than Parkin + / + MEFs under both normoxic and hypoxic conditions. h Myc-Parkin expression reduced the levels of endogenous HIF-1α protein in both VHL-deficient RCC4 cells and VHL-proficient RCC4/VHL cells. i Knockdown of Parkin increased HIF-1α protein levels in both RCC4 and RCC4/VHL cells. j Knockdown of Parkin in MCF7 cells with VHL knockdown further increased HIF-1α protein levels

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, shRNA

    Parkin promotes HIF-1α protein degradation through ubiquitination. a Proteasome inhibitor MG132 inhibited the downregulation of HIF-1α protein levels induced by Myc-Parkin expression in MCF7 and MDA-MB231 cells. b Myc-Parkin expression decreased HIF-1α protein half-life in cells. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with the HA-HIF-1α vector. The cells were treated with 50 µg ml −1 CHX for indicated time periods before being collected for western-blot assays. c Knockdown of endogenous Parkin increased HIF-1α protein half-life in MCF7 cells. In b , c , the data present mean ± S.D. ( n = 3). d The effects of expression of Myc-Parkin and its mutants on ubiquitination of HA-HIF-1α and autoubiquitination of Parkin in MCF7 cells analyzed by in vivo ubiquitination assays. e Knockdown of endogenous Parkin decreased the ubiquitination of HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. f Parkin promoted HIF-1α ubiquitination as detected by in vitro ubiquitination assays performed by incubating purified GST-Parkin and His-Trx-HIF-1α proteins in the presence of recombinant E1, E2, ubiquitin (Ub) and PINK1 proteins in vitro. g S65A Ub largely abolished ubiquitination of HIF-1α by Parkin in vitro. In vitro ubiquitination assays performed by incubating purified GST-Parkin and His-Trx-HIF-1α proteins in the presence of phosphorylated (Phospho) Ub or S65A Ub. h Mutations of Parkin that impaired Parkin’s ubiquitination activity impaired the ability of Myc-Parkin to degrade HA-HIF-1α protein in MCF7 and MDA-MB231 cells

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin promotes HIF-1α protein degradation through ubiquitination. a Proteasome inhibitor MG132 inhibited the downregulation of HIF-1α protein levels induced by Myc-Parkin expression in MCF7 and MDA-MB231 cells. b Myc-Parkin expression decreased HIF-1α protein half-life in cells. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with the HA-HIF-1α vector. The cells were treated with 50 µg ml −1 CHX for indicated time periods before being collected for western-blot assays. c Knockdown of endogenous Parkin increased HIF-1α protein half-life in MCF7 cells. In b , c , the data present mean ± S.D. ( n = 3). d The effects of expression of Myc-Parkin and its mutants on ubiquitination of HA-HIF-1α and autoubiquitination of Parkin in MCF7 cells analyzed by in vivo ubiquitination assays. e Knockdown of endogenous Parkin decreased the ubiquitination of HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. f Parkin promoted HIF-1α ubiquitination as detected by in vitro ubiquitination assays performed by incubating purified GST-Parkin and His-Trx-HIF-1α proteins in the presence of recombinant E1, E2, ubiquitin (Ub) and PINK1 proteins in vitro. g S65A Ub largely abolished ubiquitination of HIF-1α by Parkin in vitro. In vitro ubiquitination assays performed by incubating purified GST-Parkin and His-Trx-HIF-1α proteins in the presence of phosphorylated (Phospho) Ub or S65A Ub. h Mutations of Parkin that impaired Parkin’s ubiquitination activity impaired the ability of Myc-Parkin to degrade HA-HIF-1α protein in MCF7 and MDA-MB231 cells

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Western Blot, In Vivo, In Vitro, Purification, Recombinant, Activity Assay

    Parkin negatively regulates HIF-1α transcriptional activity. a Myc-Parkin expression inhibited HIF-1α luciferase reporter activities in cells. b Parkin knockdown increased HIF-1α luciferase reporter activities in cells. c Myc-Parkin expression reduced mRNA expression of HIF-1α target genes in cells. d Parkin knockdown increased mRNA expression of HIF-1α target genes in cells. Gene expression was measured by quantitative Taqman real-time PCR and normalized with actin . In c , HIF-1α was knocked down by 2 different shRNA vectors, and in d , HIF-1α and Parkin were knocked down by 2 different shRNA vectors. Similar results were observed for 2 different HIF-1α and Parkin shRNA vectors, and only results from one shRNA vector were presented for the sake of clarity. The data present mean ± S.D. ( n = 6). ** P

    Journal: Nature Communications

    Article Title: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression

    doi: 10.1038/s41467-017-01947-w

    Figure Lengend Snippet: Parkin negatively regulates HIF-1α transcriptional activity. a Myc-Parkin expression inhibited HIF-1α luciferase reporter activities in cells. b Parkin knockdown increased HIF-1α luciferase reporter activities in cells. c Myc-Parkin expression reduced mRNA expression of HIF-1α target genes in cells. d Parkin knockdown increased mRNA expression of HIF-1α target genes in cells. Gene expression was measured by quantitative Taqman real-time PCR and normalized with actin . In c , HIF-1α was knocked down by 2 different shRNA vectors, and in d , HIF-1α and Parkin were knocked down by 2 different shRNA vectors. Similar results were observed for 2 different HIF-1α and Parkin shRNA vectors, and only results from one shRNA vector were presented for the sake of clarity. The data present mean ± S.D. ( n = 6). ** P

    Article Snippet: For the co-IP of endogenous Parkin and HIF-1α, the anti-Parkin antibody (#4211, Cell signaling, 5 µg) and anti-HIF-1α antibody (sc-10790, Santa Cruz, 5 µg) were used, respectively.

    Techniques: Activity Assay, Expressing, Luciferase, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation

    Hypoxia induces HIF-1α and VEGF-A expression in ECs. (A) The hypoxic area in human tumor xenografts in nude mice was analyzed using the hypoxia marker pimonidazole and CA IX. Tumor tissues were double-stained with anti-CD31 (red) and anti-pimonidazole antibodies or anti-CA IX (green) to visualize hypoxic areas. Pimonidazole staining revealed that tumor vessels were exposed to hypoxia to some extent. Scale bar, 100 μm. (B) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. (C) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. The experiment was repeated three times. Representative data is shown. (D) mRNA levels of VEGF-A were significantly increased by hypoxia in HMVECs. Experiments were performed in triplicate. *p

    Journal: PLoS ONE

    Article Title: Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    doi: 10.1371/journal.pone.0080349

    Figure Lengend Snippet: Hypoxia induces HIF-1α and VEGF-A expression in ECs. (A) The hypoxic area in human tumor xenografts in nude mice was analyzed using the hypoxia marker pimonidazole and CA IX. Tumor tissues were double-stained with anti-CD31 (red) and anti-pimonidazole antibodies or anti-CA IX (green) to visualize hypoxic areas. Pimonidazole staining revealed that tumor vessels were exposed to hypoxia to some extent. Scale bar, 100 μm. (B) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. (C) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. The experiment was repeated three times. Representative data is shown. (D) mRNA levels of VEGF-A were significantly increased by hypoxia in HMVECs. Experiments were performed in triplicate. *p

    Article Snippet: Antibodies The following antibodies were used: FITC–anti-mouse CD31, anti-mouse (BD Pharmingen, San Diego, CA), anti-HIF-1α (Cell Signaling Technology, Danvers, MA), anti-human carbonic anhydrase ix (CA ix; R & D Systems, Minneapolis, MN), anti-HIF-1α (Cayman), and monoclonal anti-β-actin (Cell Signaling), anti-Ki67 (abcam, Cambridge, MA)

    Techniques: Expressing, Mouse Assay, Marker, Staining, Cell Culture, Western Blot

    Immunohistochemical stains of in vivo human brain tumors in nude mice. Immunohistochemical staining with antibodies against vascular endothelial growth factor (VEGF), CD31, Ephrin type-A receptor 2 (EphA2), and hypoxia-inducible factor 1α (HIF1α) was performed. The expression of VEGF and CD31 were down-regulated in PP2-treated tumors relative to those with XRT alone or XRT plus temozolomide (TMZ) tumors, suggesting that PP2 may suppress angiogenesis in in vivo tumor as well as in in vitro cells. Expression of EphA2 and HIF1α were not influenced by PP2 treatment in in vivo tumors. XRT, radiotherapy.

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: The Effect of Chemoradiotherapy with SRC Tyrosine Kinase Inhibitor, PP2 and Temozolomide on Malignant Glioma Cells In Vitro and In Vivo

    doi: 10.4143/crt.2014.320

    Figure Lengend Snippet: Immunohistochemical stains of in vivo human brain tumors in nude mice. Immunohistochemical staining with antibodies against vascular endothelial growth factor (VEGF), CD31, Ephrin type-A receptor 2 (EphA2), and hypoxia-inducible factor 1α (HIF1α) was performed. The expression of VEGF and CD31 were down-regulated in PP2-treated tumors relative to those with XRT alone or XRT plus temozolomide (TMZ) tumors, suggesting that PP2 may suppress angiogenesis in in vivo tumor as well as in in vitro cells. Expression of EphA2 and HIF1α were not influenced by PP2 treatment in in vivo tumors. XRT, radiotherapy.

    Article Snippet: Slides were incubated overnight with primary antibody against VEGF, CD31, EphA2, and HIF1α (Cell Signaling Technology, Danvers, MA), followed by incubation with secondary Alexa Fluor 488-conjugated donkey anti-goat antibody (Molecular Probes, Eugene, OR) for 1 hour.

    Techniques: Immunohistochemistry, In Vivo, Mouse Assay, Staining, Expressing, In Vitro

    Wheatgrass inhibits the hypoxia-mediated EMT process by inhibiting the activation of HIF-1α in airway epithelial cells. (A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h, and nuclear protein fractions were analyzed by immunoblotting with an anti-HIF-1α antibody. (B and C) Cells were transfected with either si-HIF-1α, si-negative control (NC), wheatgrass (150 µg/mL), or hypoxia only as indicated. E-cadherin expression levels (green) were analyzed by immunofluorescence analysis. (D and E) HIF-1α expression levels (red) were visualized by immunofluorescence analysis ( * P

    Journal: Nutrition Research and Practice

    Article Title: Wheatgrass extract inhibits hypoxia-inducible factor-1-mediated epithelial-mesenchymal transition in A549 cells

    doi: 10.4162/nrp.2017.11.2.83

    Figure Lengend Snippet: Wheatgrass inhibits the hypoxia-mediated EMT process by inhibiting the activation of HIF-1α in airway epithelial cells. (A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h, and nuclear protein fractions were analyzed by immunoblotting with an anti-HIF-1α antibody. (B and C) Cells were transfected with either si-HIF-1α, si-negative control (NC), wheatgrass (150 µg/mL), or hypoxia only as indicated. E-cadherin expression levels (green) were analyzed by immunofluorescence analysis. (D and E) HIF-1α expression levels (red) were visualized by immunofluorescence analysis ( * P

    Article Snippet: After blocking with TBS-T (20 mM Tris, 500 nM NaCl, and 0.1% Tween-20) containing 5% (w/v) skim milk, the membrane was incubated with a specific primary antibody for E-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), N-cadherin, Snail, Phospho-Smad3, Smad3 , Lamin (Cell Signaling Technology, Danvers, MA, USA), HIF-1α (Novus, Littleton, CO, USA), or GAPDH (Santa Cruz Biotechnology) followed by peroxidase-conjugated anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, USA).

    Techniques: Activation Assay, Transfection, Negative Control, Expressing, Immunofluorescence

    Smad3 association with the −335 HRE of the COL1A2 promoter is modulated by HIF-1α. For each DAPA, nuclear lysates from cells treated with 1 ng/ml TGF-β or vehicle for 6 h were used, and a representative of 3 experiments is shown.

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: Smad3 association with the −335 HRE of the COL1A2 promoter is modulated by HIF-1α. For each DAPA, nuclear lysates from cells treated with 1 ng/ml TGF-β or vehicle for 6 h were used, and a representative of 3 experiments is shown.

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques:

    TGF-β increases activity of the HIF-1α N-TAD. Cells were transfected and treated with 1-2 ng/ml TGF-β or vehicle for 24 h. In (a) and (b) constructs containing the yeast Gal4 binding domain conjugated to the specified regions of

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: TGF-β increases activity of the HIF-1α N-TAD. Cells were transfected and treated with 1-2 ng/ml TGF-β or vehicle for 24 h. In (a) and (b) constructs containing the yeast Gal4 binding domain conjugated to the specified regions of

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques: Activity Assay, Transfection, Construct, Binding Assay

    HIF-1α deletion is protective in the NEP25 model of glomerulosclerosis. (a) Formalin-fixed paraffin-embedded kidney sections were PAS stained. Representative images are shown from the indicated treatment group. Scale bar=25 μm. (b) Glomeruli

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: HIF-1α deletion is protective in the NEP25 model of glomerulosclerosis. (a) Formalin-fixed paraffin-embedded kidney sections were PAS stained. Representative images are shown from the indicated treatment group. Scale bar=25 μm. (b) Glomeruli

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques: Formalin-fixed Paraffin-Embedded, Staining

    HIF-1α enhances COL1A2 promoter induction by TGF-β. Cells were transfected and treated with 1-2 ng/ml TGF-β or vehicle control for 24 h. (a) THMC and (b) HKC expressing either control or HIF-1α shRNA were transfected with

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: HIF-1α enhances COL1A2 promoter induction by TGF-β. Cells were transfected and treated with 1-2 ng/ml TGF-β or vehicle control for 24 h. (a) THMC and (b) HKC expressing either control or HIF-1α shRNA were transfected with

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques: Transfection, Expressing, shRNA

    The −335 HRE is a functional HIF-1α-binding site. (a) The COL1A2 -luc construct was mutated at each putative HRE separately (−173, −221, or −335), or at all three, and transfected into HMC or HKC. Cells were treated

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: The −335 HRE is a functional HIF-1α-binding site. (a) The COL1A2 -luc construct was mutated at each putative HRE separately (−173, −221, or −335), or at all three, and transfected into HMC or HKC. Cells were treated

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques: Functional Assay, Binding Assay, Construct, Transfection

    TGF-β enhances HIF-1α association with −335 HRE of the COL1A2 promoter. (a) Cytoplasmic and nuclear lysates of HMC treated with 100 μM DFO and 2 ng/ml TGF-β or vehicle for 6 h. (b) Average nuclear HIF-1α

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: TGF-β enhances HIF-1α association with −335 HRE of the COL1A2 promoter. (a) Cytoplasmic and nuclear lysates of HMC treated with 100 μM DFO and 2 ng/ml TGF-β or vehicle for 6 h. (b) Average nuclear HIF-1α

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques:

    HIF-1α expression in the NEP25 model. (a) Immunohistochemistry for glomerular HIF-1α expression in healthy control and LMB2-treated WT and HIF-1α −/− NEP25 mice. Representative glomeruli are shown. Scale bar=25 μm.

    Journal: Kidney international

    Article Title: Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3

    doi: 10.1016/j.kint.2016.05.026

    Figure Lengend Snippet: HIF-1α expression in the NEP25 model. (a) Immunohistochemistry for glomerular HIF-1α expression in healthy control and LMB2-treated WT and HIF-1α −/− NEP25 mice. Representative glomeruli are shown. Scale bar=25 μm.

    Article Snippet: Formalin-fixed, paraffin-embedded 4-μm kidney sections were Periodic acid-Schiff (PAS) or silver stained, or probed using HIF-1α (Cayman) and αSMA (Abcam, Cambridge, MA) antibodies by the Mouse Histology and Phenotyping Laboratory of Northwestern University.

    Techniques: Expressing, Immunohistochemistry, Mouse Assay

    Effect of HIF-1α silencing on the hypoxia-induced protection against the etoposide-induced apoptosis. A , cells were transfected with 5, 20 or 50 nM HIF-1α siRNA, 50 nM non-targeting siRNA or with the transfection reagent alone (DMF) for 24 hours. Cells were then incubated under normoxia or hypoxia for 6 hours and total cell extracts were analyzed by western blot for HIF-1α protein level. A, B, C , cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. B , after the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction. C , caspase 3 activity was assayed. Results are expressed as means ± 1 SD (n = 3). *** p

    Journal: Molecular Cancer

    Article Title: Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity

    doi: 10.1186/1476-4598-7-27

    Figure Lengend Snippet: Effect of HIF-1α silencing on the hypoxia-induced protection against the etoposide-induced apoptosis. A , cells were transfected with 5, 20 or 50 nM HIF-1α siRNA, 50 nM non-targeting siRNA or with the transfection reagent alone (DMF) for 24 hours. Cells were then incubated under normoxia or hypoxia for 6 hours and total cell extracts were analyzed by western blot for HIF-1α protein level. A, B, C , cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. B , after the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction. C , caspase 3 activity was assayed. Results are expressed as means ± 1 SD (n = 3). *** p

    Article Snippet: Primary antibodies were as follows: mouse anti-p53 (#05-224 Upstate), mouse anti-phospho serine 15-p53 (#92865 Cell Signaling), rabbit anti-c-jun (SC-1694 Santa Cruz), rabbit anti-phospho serine 63-c-jun (#92615 Cell Signaling), rabbit anti-c-fos (SC-052 Santa Cruz), mouse anti-phospho serine 83-elk-1 (SC-8406 Santa-Cruz), mouse anti-phospho threonine 71-ATF2 (SC-8398 Santa Cruz), rabbit anti-phospho serine 133-CREB (#06-519 Upstate), rabbit anti-MEF-2 (SC-10794 Santa Cruz), mouse anti-c-myc (SC-42 Santa Cruz), rabbit anti-STAT-1α (SC-345 Santa Cruz), mouse anti-c-rel (SC-6955 Santa Cruz), rabbit anti-p50 (SC-7178 Santa Cruz), rabbit anti-p65 (SC-372 Santa Cruz), mouse anti-HIF-1α (#610958 BD Biosciences).

    Techniques: Transfection, Incubation, Western Blot, Amplification, SYBR Green Assay, Activity Assay

    A , Gene expression profiling in HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions after HIF-1α siRNA transfection. Cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before total RNA extraction, reverse transcription and amplification by real-time PCR in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction as the mean ± 1 SD for experimental triplicates or as the mean for experimental duplicates. ** p

    Journal: Molecular Cancer

    Article Title: Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity

    doi: 10.1186/1476-4598-7-27

    Figure Lengend Snippet: A , Gene expression profiling in HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions after HIF-1α siRNA transfection. Cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before total RNA extraction, reverse transcription and amplification by real-time PCR in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction as the mean ± 1 SD for experimental triplicates or as the mean for experimental duplicates. ** p

    Article Snippet: Primary antibodies were as follows: mouse anti-p53 (#05-224 Upstate), mouse anti-phospho serine 15-p53 (#92865 Cell Signaling), rabbit anti-c-jun (SC-1694 Santa Cruz), rabbit anti-phospho serine 63-c-jun (#92615 Cell Signaling), rabbit anti-c-fos (SC-052 Santa Cruz), mouse anti-phospho serine 83-elk-1 (SC-8406 Santa-Cruz), mouse anti-phospho threonine 71-ATF2 (SC-8398 Santa Cruz), rabbit anti-phospho serine 133-CREB (#06-519 Upstate), rabbit anti-MEF-2 (SC-10794 Santa Cruz), mouse anti-c-myc (SC-42 Santa Cruz), rabbit anti-STAT-1α (SC-345 Santa Cruz), mouse anti-c-rel (SC-6955 Santa Cruz), rabbit anti-p50 (SC-7178 Santa Cruz), rabbit anti-p65 (SC-372 Santa Cruz), mouse anti-HIF-1α (#610958 BD Biosciences).

    Techniques: Expressing, Incubation, Transfection, RNA Extraction, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Hypoxia and HIF-1α downregulates the transcription of gene encoding for the PKA regulatory subunit RIIB. a Expression of the genes encoding for PKA regulatory and catalytic subunits in GH3 cells grown under hypoxic conditions (1% O 2 for 18 h) as determined by real-time RT-PCR. Data are

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: Hypoxia and HIF-1α downregulates the transcription of gene encoding for the PKA regulatory subunit RIIB. a Expression of the genes encoding for PKA regulatory and catalytic subunits in GH3 cells grown under hypoxic conditions (1% O 2 for 18 h) as determined by real-time RT-PCR. Data are

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Expressing, Quantitative RT-PCR

    HIF-1a modulates PRKAR2B expression via Sp1 promoter binding and their expression levels are inverse correlated in acromegalic tumors. a , d Chromatin immunoprecipitation showing decreased Sp1 binding on the Prkar2b promoter in GH3 cells overexpressing HIF-1α. No DNA binding was quantified with HIF-1α. Rabbit IgG was used as control. b , e Co-immunoprecipitation experiment showing that both HIF-1α and HIF-1αR30A physically associate with Sp1. HIF-1α and Sp1 immunoprecipitates were blotted for Sp1. Proteins immunoprecipitated with rabbit IgG were used as controls. c , a Expression of PRKAR2B gene in normal pituitary glands ( n = 9) and acromegalic tumors (ACRO, n = 10). Data are PRAKR2B / TFIIB . * P = 0.006 ( U -test). d , b Western blot analysis of normal pituitary (NP) and GH-secreting tumors (ACRO) showed a significant loss of PRKAR2B protein expression, whereas HIF-1α protein levels measured in the same tumors are significantly increased in ACRO vs NP ( n = 4 per group due to scarcity of material for dual measurement). e , c Linear regression analysis of PRKAR2B transcript and HIF-1α protein levels in 14 acromegalic tumors. PRKAR2B was determined by real-time RT-PCR and values are PRKAR2B / TFIIB arbitrary units. HIF-1α protein was quantified from immunoblots (Chemidoc). Kendall’s τ = −0.663, r t = −0.480, P = 0.009.

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: HIF-1a modulates PRKAR2B expression via Sp1 promoter binding and their expression levels are inverse correlated in acromegalic tumors. a , d Chromatin immunoprecipitation showing decreased Sp1 binding on the Prkar2b promoter in GH3 cells overexpressing HIF-1α. No DNA binding was quantified with HIF-1α. Rabbit IgG was used as control. b , e Co-immunoprecipitation experiment showing that both HIF-1α and HIF-1αR30A physically associate with Sp1. HIF-1α and Sp1 immunoprecipitates were blotted for Sp1. Proteins immunoprecipitated with rabbit IgG were used as controls. c , a Expression of PRKAR2B gene in normal pituitary glands ( n = 9) and acromegalic tumors (ACRO, n = 10). Data are PRAKR2B / TFIIB . * P = 0.006 ( U -test). d , b Western blot analysis of normal pituitary (NP) and GH-secreting tumors (ACRO) showed a significant loss of PRKAR2B protein expression, whereas HIF-1α protein levels measured in the same tumors are significantly increased in ACRO vs NP ( n = 4 per group due to scarcity of material for dual measurement). e , c Linear regression analysis of PRKAR2B transcript and HIF-1α protein levels in 14 acromegalic tumors. PRKAR2B was determined by real-time RT-PCR and values are PRKAR2B / TFIIB arbitrary units. HIF-1α protein was quantified from immunoblots (Chemidoc). Kendall’s τ = −0.663, r t = −0.480, P = 0.009.

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Western Blot, Quantitative RT-PCR

    HIF-1 is upregulated in acromegalic pituitary tumors (ACRO) compared with the normal anterior pituitary gland (NP). a HIF-1α immunoreactivity in representative normal pituitary gland and acromegalic tumor. Signal is visualized with diaminobenzidine (DAB) staining (brown nuclei). Counterstaining with toluidine blue (blue nuclei). The graph shows the distribution of the HIF-1α immunoreactivity score on normal pituitary glands ( n = 5) and acromegalic tumors ( n = 39). The absolute numbers of samples are denoted in the graph bars. b Representative immunoblot for HIF-1α on normal pituitary glands ( n = 3) and acromegalic tumors ( n = 6). c Quantification of HIF-1α signal as determined by western blot on 5 normal pituitaries and 25 acromegalic tumors. Values are HIF-1α to β-actin ratio and presented as fold increase versus the mean normal pituitary values. Error bars: s.d. ** P

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: HIF-1 is upregulated in acromegalic pituitary tumors (ACRO) compared with the normal anterior pituitary gland (NP). a HIF-1α immunoreactivity in representative normal pituitary gland and acromegalic tumor. Signal is visualized with diaminobenzidine (DAB) staining (brown nuclei). Counterstaining with toluidine blue (blue nuclei). The graph shows the distribution of the HIF-1α immunoreactivity score on normal pituitary glands ( n = 5) and acromegalic tumors ( n = 39). The absolute numbers of samples are denoted in the graph bars. b Representative immunoblot for HIF-1α on normal pituitary glands ( n = 3) and acromegalic tumors ( n = 6). c Quantification of HIF-1α signal as determined by western blot on 5 normal pituitaries and 25 acromegalic tumors. Values are HIF-1α to β-actin ratio and presented as fold increase versus the mean normal pituitary values. Error bars: s.d. ** P

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Staining, Western Blot

    Hypoxia and HIF-1α increase GH synthesis. a Effect of hypoxia (1% O 2 for 18 h) on GH secretion on nine human acromegalic tumors in primary cell culture. For all cell culture experiments, each GH RIA value was divided to respective cell viability count as determined by WST-1 at OD450nm. Every condition was in quadruplicates and data are means ± SEM. * P

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: Hypoxia and HIF-1α increase GH synthesis. a Effect of hypoxia (1% O 2 for 18 h) on GH secretion on nine human acromegalic tumors in primary cell culture. For all cell culture experiments, each GH RIA value was divided to respective cell viability count as determined by WST-1 at OD450nm. Every condition was in quadruplicates and data are means ± SEM. * P

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Cell Culture

    Hypoxia and HIF-1α stimulate PKA activity. The effect of ( a ) HIF-1α overexpression and ( b ) hypoxia on PKA activity as determined with a nonradioactive commercial kit (Promega). Blots show imaging of the phosphorylated peptide substrate. Data are arbitrary units presented as % of mock (empty pCMV plasmid) control or normoxia (NX). * P = 0.040 to mock and P = 0.019 to normoxia (Student’s t test). Overexpression of a dominant negative catalytically inactive PKA (pdn-PKA) abolishes the stimulatory action of hypoxia on ( c ) Gh promoter activity, ( d ) endogenous rat Gh transcription, and ( e ) GH secretion. Data are means ± SEM from two experiments and presented as percentage of fold increase to each normoxia (NX). * P

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: Hypoxia and HIF-1α stimulate PKA activity. The effect of ( a ) HIF-1α overexpression and ( b ) hypoxia on PKA activity as determined with a nonradioactive commercial kit (Promega). Blots show imaging of the phosphorylated peptide substrate. Data are arbitrary units presented as % of mock (empty pCMV plasmid) control or normoxia (NX). * P = 0.040 to mock and P = 0.019 to normoxia (Student’s t test). Overexpression of a dominant negative catalytically inactive PKA (pdn-PKA) abolishes the stimulatory action of hypoxia on ( c ) Gh promoter activity, ( d ) endogenous rat Gh transcription, and ( e ) GH secretion. Data are means ± SEM from two experiments and presented as percentage of fold increase to each normoxia (NX). * P

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Activity Assay, Over Expression, Imaging, Plasmid Preparation, Dominant Negative Mutation

    Hypoxia and HIF-1 trigger CREB activity. a Knocking down CREB with siRNA abolishes the effect of hypoxia on endogenous rat Gh transcription in GH3 cells as determined by real-time RT-PCR. Immunoblot shows the knockdown efficacy of the CREB siRNA ( b ) effect of hypoxia (1% O 2 for 18 h) on CRE induced luciferase activity. Transfection with 100 nM HIF-1α siRNA for 48 h abolished the effect of hypoxia. Luc/βGal: luciferase: β-galactosidase ratio. Data are means ± SEM of three experiments and expressed as percentage of each normoxia control. * P

    Journal: Oncogene

    Article Title: Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription

    doi: 10.1038/s41388-020-1223-6

    Figure Lengend Snippet: Hypoxia and HIF-1 trigger CREB activity. a Knocking down CREB with siRNA abolishes the effect of hypoxia on endogenous rat Gh transcription in GH3 cells as determined by real-time RT-PCR. Immunoblot shows the knockdown efficacy of the CREB siRNA ( b ) effect of hypoxia (1% O 2 for 18 h) on CRE induced luciferase activity. Transfection with 100 nM HIF-1α siRNA for 48 h abolished the effect of hypoxia. Luc/βGal: luciferase: β-galactosidase ratio. Data are means ± SEM of three experiments and expressed as percentage of each normoxia control. * P

    Article Snippet: Primary antibodies used in this study were: CREB (Cell Signaling, Cat. 86B10—western Blot, ChIP), phospho-CREB Ser133 (Cell Signaling Cat. 87G3—western Blot) Anti-FLAG M2 (Sigma, Cat. F3165—western Blot), Anti-HIF-1α (Novus, Cat. NB100134—western Blot, Immunohistochemistry, Co-Immunoprecipitation, ChIP), Pit-1 (Santa Cruz Cat. Sc-442—western Blot, ChIP), PP1a (Santa Cruz Cat. Sc-7482—western Blot), Sp1 (Santa Cruz Cat Sc59X—western Blot, ChIP), and PKA IIB Reg—H90 (Santa Cruz Cat. sc-25424).

    Techniques: Activity Assay, Quantitative RT-PCR, Luciferase, Transfection