Product Citations: 366

Fig.5.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
Fig.2.A,B,C showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
Fig.6.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
Fig.4.D showing Western Blotting from the publication: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression.
Fig.1.B showing Western Blotting from the publication: The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.
Fig.1.D showing Western Blotting from the publication: The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.
Fig.5.B showing Western Blotting from the publication: A HIF-1α inhibitor combined with palmitic acid and L-carnitine treatment can prevent the fat metabolic reprogramming under hypoxia and induce apoptosis in hepatocellular carcinoma cells.
Fig.1.C showing Immunohistochemistry in a Homo sapiens (Human) sample from the publication: Cytoplasmic HIF-2α as tissue biomarker to identify metastatic sympathetic paraganglioma.
Fig.3.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.
Fig.3.A showing Western Blotting from the publication: FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.
Fig.1.A showing Western Blotting from the publication: FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.
Fig.1.A showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
Fig.1.D showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
Fig.3.C showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
Fig.9.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells.
Fig.6.H showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
Fig.5.G showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
Fig.1.I showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
Fig.1.K showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
Fig.1.B showing Western Blotting from the publication: p53 Is Regulated in a Biphasic Manner in Hypoxic Human Papillomavirus Type 16 (HPV16)-Positive Cervical Cancer Cells.
Fig.1.B showing Western Blotting from the publication: Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity.
Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Signs of Chronic Hypoxia Suggest a Novel Pathophysiological Event in α-Synucleinopathies.
Fig.5.A showing Western Blotting from the publication: Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.
Fig.5.C showing Western Blotting from the publication: Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.
123 images found

Targeted degradation of α-synuclein prevents PFF-induced aggregation

Preprint on BioRxiv : the Preprint Server for Biology on 19 April 2025 by Carton, B., Gelders, G., et al.

Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Therefore, clearance of misfolded α-synuclein from dopaminergic neurons could in principle offer a therapeutic window for Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein prevents the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach represents the first demonstration of nanobody-guided proteasomal degradation of all clinically relevant α-synuclein variants, highlighting its potential as a therapeutic strategy against Synucleinopathies.

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Iron content of glioblastoma tumours and role of ferrous iron in the hypoxic response in vitro.

In Frontiers in Oncology on 24 March 2025 by Praditi, C., Beverley-Stone, E., et al.

Glioblastomas are an aggressive primary brain cancer, characterised by hypoxia and poor patient survival. Iron is the most abundant transition metal in the brain, yet data on the iron content of brain cancers is sparse. Ferrous iron is an essential cofactor for a super-family of enzymes, the iron- and 2-oxoglutarate-dependent dioxygenase enzymes (2-OGDD). These enzymes control the response to hypoxia via hydroxylation of the hypoxia-inducible factor-1α (HIF-1α), and DNA demethylation via hydroxylation of 5-methyl cytosines (5hmC).
This study used clinical glioblastoma samples from 40 patients to determine the relationship between 2-OGDD activity and iron. Elemental iron was measured using inductively coupled plasma mass spectrometry (ICP-MS) and ferrous iron was measured using the colorimetric ferrozine assay. Iron measurements were compared against patient survival and clinicopathological data, and 2-OGDD-dependent activity of HIF-1 activation and 5hmC.
Elemental and ferrous iron levels were weakly related. Higher ferrous iron content of clinical glioblastoma tissue was associated with longer overall survival compared to lower ferrous iron content, but elemental iron showed no such relationship. Neither form of iron was related to clinicopathological data or markers of 2-OGDD activity. The impact of iron supplementation on the hypoxic response was assessed in three glioblastoma cell lines in vitro, similarly showing only a limited influence of iron on these 2-OGDD enzymes. Our data, together with prior studies in anaemic patients, highlight the importance of healthy iron levels in patients with glioblastoma, but further mechanistic studies are needed to elucidate the molecular pathways involved.
Copyright © 2025 Praditi, Beverley-Stone, Reid, Burgess, Crake, Vissers, Royds, Slatter, Dachs and Phillips.

  • WB
  • Homo sapiens (Human)
  • Cancer Research
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The mediator subunit complex protein MED15 promotes lipid deposition and cancer progression during hypoxia.

In The Journal of Biological Chemistry on 1 March 2025 by Zhang, B., Zhu, Y., et al.

Hypoxia, a hallmark of solid tumors, is associated with increased lipid droplet (LD) accumulation. However, the mechanisms underlying this remain elusive. Here, we identify Mediator complex subunit 15 (MED15) as a critical regulator of hypoxia-inducible factor (HIF) signaling, potentially impacting LD accumulation. In mammalian cells, we elucidated that MED15, as a HIF target gene, participates in promoting HIF transcriptional activity without affecting HIFα protein levels, creating a positive feedback loop. Furthermore, zebrafish deficiency in med15 displayed decreased HIF activity and impaired tolerance to hypoxic stress. Functionally, MED15 deficiency attenuated the proliferation of colon and renal cancer cells in vitro and tumor growth in vivo. Mechanistically, MED15 acts upstream of carnitine palmitoyltransferase 1A (CPT1A), a key enzyme in fatty acid oxidation, ultimately promoting HIF-mediated LD accumulation. Disrupting the MED15-CPT1A axis impairs this process. These findings reveal a novel MED15-HIF-CPT1A axis that promotes LD formation, potentially contributing to hypoxic tumor progression.
Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.

  • Biochemistry and Molecular biology
  • Cancer Research
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Loss-of-function mutations in the dystonia gene THAP1 impair proteasome function by inhibiting PSMB5 expression.

In Nature Communications on 10 February 2025 by Ramage, D. E., Grant, D. W., et al.

The 26S proteasome is a multi-catalytic protease that serves as the endpoint for protein degradation via the ubiquitin-proteasome system. Proteasome function requires the concerted activity of 33 distinct gene products, but how the expression of proteasome subunits is regulated in mammalian cells remains poorly understood. Leveraging coessentiality data from the DepMap project, here we characterize an essential role for the dystonia gene THAP1 in maintaining the basal expression of PSMB5. PSMB5 insufficiency resulting from loss of THAP1 leads to defects in proteasome assembly, impaired proteostasis and cell death. Exploiting the fact that the toxicity associated with loss of THAP1 can be rescued upon exogenous expression of PSMB5, we define the transcriptional targets of THAP1 through RNA-seq analysis and perform a deep mutational scan to systematically assess the function of thousands of single amino acid THAP1 variants. Altogether, these data identify THAP1 as a critical regulator of proteasome function and suggest that aberrant proteostasis may contribute to the pathogenesis of THAP1 dystonia.
© 2025. The Author(s).

  • Homo sapiens (Human)
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Hypoxia-induced translation of collagen-modifying enzymes PLOD2 and P4HA1 is dependent on RBM4 and eIF4E2 in human colon cancer HCT116 cells.

In The FEBS Journal on 1 February 2025 by Li, H. H., Hung, H. Y., et al.

Hypoxia is a critical microenvironmental factor that induces tumorigenesis and cancer progression, including metastasis. The highly dynamic nature of the extracellular matrix (ECM) plays a crucial role in metastasis. Collagens are the predominant component of structural proteins embedded within the ECM. The biosynthesis of collagen typically undergoes a series of posttranslational modifications, such as hydroxylation of lysine and proline residues by procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) and prolyl 4-hydroxylases (P4Hs), respectively. Collagen hydroxylation is critical for ECM remodeling and maintenance. We recently investigated hypoxia-induced translation in human colon cancer HCT116 cells and identified several collagen-modifying enzymes, including procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and prolyl 4-hydroxylase subunit alpha 1 (P4HA1). Although the translation of bulk mRNAs is repressed in hypoxia, specific mRNAs remain efficiently translated under such conditions. We have found that PLOD2 and P4HA1 are significantly upregulated in hypoxic HCT116 cells compared to normoxic cells. HIF-1 is known to induce the transcription of PLOD2 and P4HA1 during hypoxia. However, the molecular mechanisms of hypoxia-induced translation of PLOD2 and P4HA1 remain largely unclear. We provide evidence that RBM4 and eIF4E2 are required for hypoxia-induced translation of PLOD2 and P4HA1 mRNAs. The 3' UTRs of PLOD2 and P4HA1 mRNAs are involved in translational control during hypoxia in HCT116 cells.
© 2024 Federation of European Biochemical Societies.

  • WB
  • Biochemistry and Molecular biology
  • Cancer Research
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The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.

In Cell Mol Biol Lett on 8 July 2024 by Peng, S., Guo, Y., et al.

Fig.5.F
Fig.5.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
  • WB
  • Homo sapiens (Human)
Docetaxel (DTX) and Yard. 2 exhibit similar but distinct actions. A and B PC3 cells were incubated in normoxia (Nx) for 24, 48h, and 72h in the absence (Ctl) or presence of 10, 30 or 100nM of Docetaxel (DTX). Cell proliferation (A) and cell viability (B) were measured using an ADAM cell counter. ... more
Docetaxel (DTX) and Yard. 2 exhibit similar but distinct actions. A and B PC3 cells were incubated in normoxia (Nx) for 24, 48h, and 72h in the absence (Ctl) or presence of 10, 30 or 100nM of Docetaxel (DTX). Cell proliferation (A) and cell viability (B) were measured using an ADAM cell counter. C and D PC3 cells were incubated in hypoxia (Hx 1%) for 24, 48h, and 72h in the absence (Ctl) or presence of 10, 30 or 100nM of Docetaxel (DTX). Cell proliferation (C) and cell viability (D) were measured using an ADAM cell counter. A–D * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0005. Data represent the mean ± SD of experiments performed at least three times. E Cell cycle effect of PC3 cells treated with 30nM of DTX. F PC3 cells were treated with 10 or 30nM of Docetaxel (DTX) and subjected to hypoxia 1% O2 (Hx 1%) for 48h. Cell lysates were analyzed by immunoblotting for HIF-1α. β-Actin was used as a loading control. G PC3 cells were treated in hypoxia (Hx 1%) in the presence of Yard. 2 at 20 μM and in the presence of Docetaxel (DTX) at 30nM for 48h. Venn diagram showing the differential distribution of the genes detected between Hx + Yard. 2 and Hx (in blue) and between Hx + DTX and Hx (in red). Gene set enrichment list of RNA-Seq data using “Cellular functions” less

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The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.

In Cell Mol Biol Lett on 8 July 2024 by Peng, S., Guo, Y., et al.

Fig.2.A,B,C
Fig.2.A,B,C showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
  • WB
  • Homo sapiens (Human)
Effect of Sodwanones and Yardenones on HIF-1α stabilization. A P69, DU145, PC3, and 786-O cells were subjected to hypoxia 1% O2 (Hx 1%) for 24 and 48h. Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. B P69, DU145, and PC3 cells were treated with Sod... more
Effect of Sodwanones and Yardenones on HIF-1α stabilization. A P69, DU145, PC3, and 786-O cells were subjected to hypoxia 1% O2 (Hx 1%) for 24 and 48h. Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. B P69, DU145, and PC3 cells were treated with Sodwanone A (Sod. A) at 20 μM for 48h in hypoxia (Hx 1%). Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. C, P69, DU145, and PC3 cells were treated with Yardenone 2 (Yard. 2) at 20 μM for 48h in hypoxia (Hx 1%). Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. D, Immunofluorescence labeling and merge images showing the nuclear localization of HIF-1α (in green) and DAPI (in blue) in PC3 cells treated with Sod. A and Yard. 2 at 20 μM for 48h in hypoxia (Hx 1%) less

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The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.

In Cell Mol Biol Lett on 8 July 2024 by Peng, S., Guo, Y., et al.

Fig.6.B
Fig.6.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes.
  • WB
  • Homo sapiens (Human)
Yard. 2 directly targets HIF-1α, subsequently inhibiting the induction of genes that later impact tubulin. A Immunofluorescence labeling and merged images with HIF-1α (in green), α-tubulin (in red), and DAPI (in blue) for PC3 cells treated with 30 nM of Docetaxel (DTX) or 20 µM of Yard. 2 and inc... more
Yard. 2 directly targets HIF-1α, subsequently inhibiting the induction of genes that later impact tubulin. A Immunofluorescence labeling and merged images with HIF-1α (in green), α-tubulin (in red), and DAPI (in blue) for PC3 cells treated with 30 nM of Docetaxel (DTX) or 20 µM of Yard. 2 and incubated for 48 h in Hx. The yellow arrow indicates condensed tubulin, while the green arrow signifies mitotic catastrophe. B PC3 cells were stably transfected with the inducible (doxycycline) pLKO-TetOn-Puromycin encoding shRNA to HIF-1α. PC3 expressing shRNA HIF-1α were subjected to hypoxia (Hx1%) in the absence (−) or presence ( +) of doxycycline. Cell lysates were analyzed by immunoblotting for HIF-1α. β-Actin was used as a loading control. C Graphic representation of HIF-1α, Ca9 and Glut1 mRNA expression in PC3 cells incubated in hypoxia (Hx—1% O2) %) in the absence (−) or presence ( +) of doxycycline for 48 h. The one-way ANOVA is representative of two independent experiments. D Immunofluorescence labeling and merged images with HIF-1α (in green), α-tubulin (in red), and DAPI (in blue) for PC3 expressing shRNA HIF-1α in the absence (−) or in the presence ( +) of doxycycline and incubated for 48 h in Hx. E Graph illustrating the comparison of the modes of action of Yard. 2 versus Docetaxel less

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MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression.

In Cell Stress on 13 March 2024 by Lin, Z., Roche, M. E., et al.

Fig.4.D
Fig.4.D showing Western Blotting from the publication: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression.
  • WB
FIGURE 4: Effect of miR-200c overexpression in fibroblasts on NFκB and HIF pathways.(A) Phospho-NFκB p65 expression of fibroblasts with miR-200c downregulation or overexpression. (B) NFκB activity in NIH3T3 NFκB luciferase reporter cells with miR-200c overexpression or downregulation under normox... more
FIGURE 4: Effect of miR-200c overexpression in fibroblasts on NFκB and HIF pathways.(A) Phospho-NFκB p65 expression of fibroblasts with miR-200c downregulation or overexpression. (B) NFκB activity in NIH3T3 NFκB luciferase reporter cells with miR-200c overexpression or downregulation under normoxic (21% O2) and hypoxic (1% O2) conditions (Random Luciferase Units-RLU). *p<0.05. (C) Cytokine, chemokine and receptor expression profile in fibroblast conditioned media. BJ1 F200c or control were cultured in serum-free DMEM media. The levels of various factors in the cell-free culture media were measured by antibody arrays at day 2 and 4. Data was showed as fold change ± SD. *p<0.05. (D) HIF and MCT4 expression in fibroblasts with miR-200c modulation. (E) HIF activity in NIH3T3 HIF luciferase reporter cells with miR-200c overexpression or downregulation under normoxic and hypoxic conditions. *p<0.05. (F) Lactate level in BJ1miR-200c and control under normoxic and hypoxic conditions. *p<0.05. less

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The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.

In Adv Sci (Weinh) on 1 February 2024 by Wang, J., Yang, C., et al.

Fig.1.B
Fig.1.B showing Western Blotting from the publication: The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.
  • WB
EZH2 mediates resistance to HIF‐1 inhibitors in lung cancer cells. A) Screening for HIF‐1 inhibitor‐resistant cells using an epigenetic compound library. B) HIF‐1 inhibitors promote high expression of EZH2 substrate H3K27me3 in A549 and H460 cell lines. C) Under normoxia and hypoxia, the knockdow... more
EZH2 mediates resistance to HIF‐1 inhibitors in lung cancer cells. A) Screening for HIF‐1 inhibitor‐resistant cells using an epigenetic compound library. B) HIF‐1 inhibitors promote high expression of EZH2 substrate H3K27me3 in A549 and H460 cell lines. C) Under normoxia and hypoxia, the knockdown of HIF‐1α promotes high expression of the EZH2 substrate H3K27me3. D) Rescuing HIF‐1α reduces H3K27me3 protein levels in A549 and H460 HIF‐1α knockout cells. E,F) Knockout of HIF‐1α promotes protein and mRNA levels of SUZ12. **p < 0.01,***p < 0.001, as compared with the V2 group. Rescuing HIF‐1α reduces SUZ12 mRNA levels G) and protein levels H) in HIF‐1α knockout cells. **p < 0.01, ***p < 0.001, as compared with the pcDNA group. less

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The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.

In Adv Sci (Weinh) on 1 February 2024 by Wang, J., Yang, C., et al.

Fig.1.D
Fig.1.D showing Western Blotting from the publication: The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.
  • WB
EZH2 mediates resistance to HIF‐1 inhibitors in lung cancer cells. A) Screening for HIF‐1 inhibitor‐resistant cells using an epigenetic compound library. B) HIF‐1 inhibitors promote high expression of EZH2 substrate H3K27me3 in A549 and H460 cell lines. C) Under normoxia and hypoxia, the knockdow... more
EZH2 mediates resistance to HIF‐1 inhibitors in lung cancer cells. A) Screening for HIF‐1 inhibitor‐resistant cells using an epigenetic compound library. B) HIF‐1 inhibitors promote high expression of EZH2 substrate H3K27me3 in A549 and H460 cell lines. C) Under normoxia and hypoxia, the knockdown of HIF‐1α promotes high expression of the EZH2 substrate H3K27me3. D) Rescuing HIF‐1α reduces H3K27me3 protein levels in A549 and H460 HIF‐1α knockout cells. E,F) Knockout of HIF‐1α promotes protein and mRNA levels of SUZ12. **p < 0.01,***p < 0.001, as compared with the V2 group. Rescuing HIF‐1α reduces SUZ12 mRNA levels G) and protein levels H) in HIF‐1α knockout cells. **p < 0.01, ***p < 0.001, as compared with the pcDNA group. less

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A HIF-1α inhibitor combined with palmitic acid and L-carnitine treatment can prevent the fat metabolic reprogramming under hypoxia and induce apoptosis in hepatocellular carcinoma cells.

In Cancer Metab on 8 December 2023 by Matsufuji, S., Kitajima, Y., et al.

Fig.5.B
Fig.5.B showing Western Blotting from the publication: A HIF-1α inhibitor combined with palmitic acid and L-carnitine treatment can prevent the fat metabolic reprogramming under hypoxia and induce apoptosis in hepatocellular carcinoma cells.
  • WB
Regulation of fatty acid metabolism-related genes by HIF-1α under hypoxia. A qRT-PCR analysis of mRNA expression levels of genes encoding fatty acid β-oxidation (FAO)-related enzymes ACSL1, CPT1, MCAD, and LCAD, as well as fatty acid transporter CD36, in HIF-1α knockdown (KD) and scramble control... more
Regulation of fatty acid metabolism-related genes by HIF-1α under hypoxia. A qRT-PCR analysis of mRNA expression levels of genes encoding fatty acid β-oxidation (FAO)-related enzymes ACSL1, CPT1, MCAD, and LCAD, as well as fatty acid transporter CD36, in HIF-1α knockdown (KD) and scramble control (SC) HepG2 and Hep3B cells. B Western blot analysis of ACSL1, CPT1, MCAD, LCAD, and CD36 protein expression in KD and SC cells. C Quantification of Western blot analysis of ACSL1, CPT1, MCAD, LCAD, and CD36 protein expression. D qRT-PCR analysis of mRNA levels of triglyceride (TG) synthesis-related genes APGAT and DGAT in KD and SC cells. E Western blot analysis of APGAT and DGAT protein expression in KD and SC cells. F Quantification of Western blot analysis of APGAT, DGAT protein expression. Data are expressed as the mean ± standard error of the mean (SEM) and are representative of three independent experiments (A, D). N.S.: not significant, *P < 0.05, **P < 0.01 versus control less

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Cytoplasmic HIF-2α as tissue biomarker to identify metastatic sympathetic paraganglioma.

In Sci Rep on 18 July 2023 by Karakaya, S., Gunnesson, L., et al.

Fig.1.C
Fig.1.C showing Immunohistochemistry in a Homo sapiens (Human) sample from the publication: Cytoplasmic HIF-2α as tissue biomarker to identify metastatic sympathetic paraganglioma.
  • IHC
  • Homo sapiens (Human)
PPGLs express low levels of Ki-67 and HIF-1α. (A) Representative images from tumor cores scored as negative (0), weak (1 +), intermediate (2 +) or strong (3 +) for Ki67. (B) Quantification of Ki67 positive cells (in %). (C) Representative images from tumor cores scored as negative (0), weak (1 +)... more
PPGLs express low levels of Ki-67 and HIF-1α. (A) Representative images from tumor cores scored as negative (0), weak (1 +), intermediate (2 +) or strong (3 +) for Ki67. (B) Quantification of Ki67 positive cells (in %). (C) Representative images from tumor cores scored as negative (0), weak (1 +), intermediate (2 +) or strong (3 +) for HIF-1α. (D) Quantification of HIF-1α positive cells (in %). Tumor cores were scored manually by three independent researchers as well as semi-automatically using the QuPath-0.3.0 software. Statistical significance was determined by the Mann–Whitney test. sPGL, sympathetic paraganglioma; HN-PGL, head- and neck paraganglioma; PCC, pheochromocytoma. The scale bar represents 100 μm in core images and 50 μm in zoomed images. less

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The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.

In Cell Death Dis on 10 June 2023 by Domingo-Reinés, J., Montes, R., et al.

Fig.3.E
Fig.3.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.
  • WB
  • Homo sapiens (Human)
Transcriptomic analysis of NUP98-KDM5A expressing-iPSC.A Principal Component Analysis for RNA-seq data in iPSC-control and iPSC-NK5A clones. B Hierarchical clustering heat map of the differentially expressed genes (fold change log2 > 1 or <−1 and adjusted p value < 0.05) between iPSC-control and ... more
Transcriptomic analysis of NUP98-KDM5A expressing-iPSC.A Principal Component Analysis for RNA-seq data in iPSC-control and iPSC-NK5A clones. B Hierarchical clustering heat map of the differentially expressed genes (fold change log2 > 1 or <−1 and adjusted p value < 0.05) between iPSC-control and iPSC-NK5A clones. C Top 13 ranked gene sets in the GSEA of genes up-regulated in NK5A expressing iPSCs compared with control samples. D Hierarchical clustering heat map of TOP8 GSEA hypoxia-related gene sets. Direct targets of HIF1A from Harmonizome data base are highlighted: Validated (green) and Predicted (orange). E Western blot analysis detecting HIF1A in iPSC-control and iPSC-NK5A clones. ACTIN is used as a loading control. F Quantification of the levels of HIF1A protein in iPSC-control and NK5A clones, n = 3. Red line in (C) is p values < 0.05. **p < 0.01; Two-tailed unpaired Student’s t-student test applied in (F). less

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FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.

In Cells on 21 October 2022 by Yang, D., Strobel, T. D., et al.

Fig.3.A
Fig.3.A showing Western Blotting from the publication: FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.
  • WB
FAM57A expression in relation to cell density, normoxia/hypoxia, and glucose supply. (a) Immunoblot analyses of HPV-positive cancer cells seeded at LD or HD. After 24 h, cells were cultivated at normoxic (21% O2) or hypoxic (1% O2) conditions and under different glucose concentrations, as indicat... more
FAM57A expression in relation to cell density, normoxia/hypoxia, and glucose supply. (a) Immunoblot analyses of HPV-positive cancer cells seeded at LD or HD. After 24 h, cells were cultivated at normoxic (21% O2) or hypoxic (1% O2) conditions and under different glucose concentrations, as indicated, for another 24 h before harvesting. HIF-1α, hypoxia marker; GAPDH, loading control. (b) qRT-PCR analyses determining the mean (log2FC) FAM57A transcript levels in HeLa-2 cells cultivated at hypoxia (H; 1% O2) compared to normoxia (N; 21% O2) (log2FC = 0) with standard deviations from 4 independent experiments. Statistically significant differences are determined by one-way ANOVA. ***: p < 0.001. (c) qRT-PCR analyses measuring FAM57A transcript levels following silencing HIF1A (siHIF1A) or HIF2A (siHIF2A) expression, either alone or in combination. Displayed are the log2FC of mean FAM57A transcript levels in normoxic (N) and hypoxic (H) cells, relative to control siRNA (siCtrl)-transfected cells cultivated at normoxia (log2FC = 0) from 3 independent experiments with standard deviations. Statistically significant differences are determined by one-way ANOVA. **: p < 0.01, ***: p < 0.001. less

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FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.

In Cells on 21 October 2022 by Yang, D., Strobel, T. D., et al.

Fig.1.A
Fig.1.A showing Western Blotting from the publication: FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.
  • WB
Comparative analyses of FAM57A protein and transcript levels in cells cultivated at normoxia or hypoxia. (a) HPV-positive cervical cancer cells (SiHa, CaSki, HeLa-1) and HaCaT keratinocytes were cultivated for 24 h at normoxia (N; 21% O2) or hypoxia (H; 1% O2) and analyzed by immunoblot for FAM57... more
Comparative analyses of FAM57A protein and transcript levels in cells cultivated at normoxia or hypoxia. (a) HPV-positive cervical cancer cells (SiHa, CaSki, HeLa-1) and HaCaT keratinocytes were cultivated for 24 h at normoxia (N; 21% O2) or hypoxia (H; 1% O2) and analyzed by immunoblot for FAM57A protein levels. HIF-1α, hypoxia marker; β-Actin and α-Tubulin, loading controls. (b) Corresponding qRT-PCR analyses of FAM57A transcript levels under the conditions outlined in subfigure (a). Shown are the log2-transformed fold changes (log2FC) of mean expression in cells cultivated at hypoxia compared to normoxia, with standard deviations from at least 3 independent experiments. Statistically significant differences in cells cultivated at hypoxia compared to normoxia (log2FC = 0) are determined by one-way ANOVA. **: p < 0.01, ***: p < 0.001. less

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Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

In MBio on 23 March 2021 by Wise, L. M., Xi, Y., et al.

Fig.1.A
Fig.1.A showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
  • WB
HIF1α is induced by HCMV infection and limits virus replication in normoxia. (A) HIF1α protein levels in uninfected and HCMV-infected fibroblasts cells were determined by Western blotting. Cells were infected at an MOI of 3 infectious units per cell with strain TB40/E when cells were 80% confluen... more
HIF1α is induced by HCMV infection and limits virus replication in normoxia. (A) HIF1α protein levels in uninfected and HCMV-infected fibroblasts cells were determined by Western blotting. Cells were infected at an MOI of 3 infectious units per cell with strain TB40/E when cells were 80% confluent. Uninfected cells continued to grow, reaching full confluence by day 1 to 2. (B) Quantification of HIF1α protein relative to tubulin in cells described for panel A. Data are means ± SD, and significance was determined by unpaired t test (n = 3) (*, P < 0.05). (C) HIF1α KO cells were generated using CRISPR/Cas9 in HFF-hTERT cells. Shown is the indel sequencing for HIF1α KO and the sequence from cells treated with a nontargeting (NT) gRNA. (D) HIF1α protein level in NT and HIF1α KO cells following 24 h treatment with 100 μM CoCl2 to mimic hypoxia. (E) HIF1α KO and NT cells were infected at an MOI of 0.05, and virus growth was measured by 50% tissue culture infective dose (TCID50) at the indicated days postinfection (dpi). Data are means ± SD, and significance was determined by ANOVA and Tukey’s test (n = 3) (**, P < 0.01) (day 6 excludes an outlier, which is shown in Fig. S1). (F) Virus replication in HIF1α KO and NT cells at an MOI of 3 (4 dpi; n = 3). less

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Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

In MBio on 23 March 2021 by Wise, L. M., Xi, Y., et al.

Fig.1.D
Fig.1.D showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
  • WB
HIF1α is induced by HCMV infection and limits virus replication in normoxia. (A) HIF1α protein levels in uninfected and HCMV-infected fibroblasts cells were determined by Western blotting. Cells were infected at an MOI of 3 infectious units per cell with strain TB40/E when cells were 80% confluen... more
HIF1α is induced by HCMV infection and limits virus replication in normoxia. (A) HIF1α protein levels in uninfected and HCMV-infected fibroblasts cells were determined by Western blotting. Cells were infected at an MOI of 3 infectious units per cell with strain TB40/E when cells were 80% confluent. Uninfected cells continued to grow, reaching full confluence by day 1 to 2. (B) Quantification of HIF1α protein relative to tubulin in cells described for panel A. Data are means ± SD, and significance was determined by unpaired t test (n = 3) (*, P < 0.05). (C) HIF1α KO cells were generated using CRISPR/Cas9 in HFF-hTERT cells. Shown is the indel sequencing for HIF1α KO and the sequence from cells treated with a nontargeting (NT) gRNA. (D) HIF1α protein level in NT and HIF1α KO cells following 24 h treatment with 100 μM CoCl2 to mimic hypoxia. (E) HIF1α KO and NT cells were infected at an MOI of 0.05, and virus growth was measured by 50% tissue culture infective dose (TCID50) at the indicated days postinfection (dpi). Data are means ± SD, and significance was determined by ANOVA and Tukey’s test (n = 3) (**, P < 0.01) (day 6 excludes an outlier, which is shown in Fig. S1). (F) Virus replication in HIF1α KO and NT cells at an MOI of 3 (4 dpi; n = 3). less

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Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

In MBio on 23 March 2021 by Wise, L. M., Xi, Y., et al.

Fig.3.C
Fig.3.C showing Western Blotting from the publication: Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.
  • WB
HIF1α suppresses kynurenine (KYN) levels in HCMV-infected cells. (A) Intracellular KYN levels in uninfected and HCMV-infected NT and HIF1α KO cells were measured by LC-MS/MS. Cells were infected at an MOI of 3, and metabolites were extracted at 2 dpi. The mean is represented by a black bar, and s... more
HIF1α suppresses kynurenine (KYN) levels in HCMV-infected cells. (A) Intracellular KYN levels in uninfected and HCMV-infected NT and HIF1α KO cells were measured by LC-MS/MS. Cells were infected at an MOI of 3, and metabolites were extracted at 2 dpi. The mean is represented by a black bar, and significance was determined by 2-way ANOVA and Tukey’s test (n ≥ 6) (*, P < 0.05; **, P < 0.01). (B) KYN levels in the extracellular environment under conditions described for part A. The mean is represented by a black bar, and significance was determined by 2-way ANOVA and Tukey’s test, n ≥ 6 (*, P < 0.05; **, P < 0.01). (C) Western blot analysis of HIF1α KO cells expressing GFP or HIF1α containing a silent mutation to mutate the Cas9 PAM recognition site. GFP and HIF1α expression was induced using a doxycycline (dox)-inducible system. Cells were treated with increasing concentrations of doxycycline for 2 days. (D) Intracellular KYN levels in HCMV-infected HIF1α KO cells expressing GFP or HIF1α-Cas9 PAM mutant. Cells were infected for 1 h at an MOI of 2 and then treated with 1 μg/ml doxycycline prior to extraction of metabolites at 2 dpi. The mean is represented by a black bar, and significance was determined using an unpaired t test (n = 3) (**, P < 0.01). less

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Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells.

In Int J Mol Sci on 1 March 2021 by Han, J. M., Sohng, J. K., et al.

Fig.9.A
Fig.9.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells.
  • WB
  • Homo sapiens (Human)
Effect of CypA knockdown on the HIF-1α and VEGF expression in AGS cells. AGS cells were transfected with CypA siRNAs (10 nM) and incubated for 24 h. The cells were incubated for 8 h under either normoxic or hypoxic conditions. (A) HIF-1α protein levels were detected by western blot analysis and f... more
Effect of CypA knockdown on the HIF-1α and VEGF expression in AGS cells. AGS cells were transfected with CypA siRNAs (10 nM) and incubated for 24 h. The cells were incubated for 8 h under either normoxic or hypoxic conditions. (A) HIF-1α protein levels were detected by western blot analysis and further quantified by densitometry. The level of β-actin was used as an internal control. (B) The concentration of VEGF protein in the supernatant was determined by a VEGF ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the hypoxic control. less

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The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.

In Nat Commun on 26 February 2021 by Zheng, F., Chen, J., et al.

Fig.6.H
Fig.6.H showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
  • WB
  • Homo sapiens (Human)
HIFAL is essential for the HIF-1α transcription activities.a The HRE element transcription activity is inhibited by HIFAL-LNA upon hypoxia. b–d HIFAL knockdown decreases the binding of HIF-1α to the promoter of LDHA (b) and PDK1 (c). HIFAL levels are shown in (d). e, f HIFAL knockout decreases th... more
HIFAL is essential for the HIF-1α transcription activities.a The HRE element transcription activity is inhibited by HIFAL-LNA upon hypoxia. b–d HIFAL knockdown decreases the binding of HIF-1α to the promoter of LDHA (b) and PDK1 (c). HIFAL levels are shown in (d). e, f HIFAL knockout decreases the binding of p300 to the promoter of LDHA (e) and the histone H3K9 acetylation on the LDHA promoter (f). Primers spanning the LDHA locus [LDHA(A)], −873~−697bp[LDHA(B)] and +631~+823 bp[(LDHA(C)] were used. g HIFAL knockout decreases the chromatin sensitivity to micrococcal nuclease. h, The binding kinetics of HIF-1α to PKM2 and PHD3 keeps increasing after hypoxia in the presence of HIFAL. HIF-1α co-immunoprecipitation (Co-IP) assay was performed at indicated time points after hypoxia and normalized to the corresponding HIF-1a protein levels. i Distribution profiles of HIF-1α binding genes flanking the transcription start sites (TSS) in HIFAL WT or null MDA-MB-231 cells under hypoxia as determined by ChIP-seq. The 10 kb regions centered on all the TSSs of HIF-1α binding genes were shown. Red indicates enrichment, while blue indicates no enrichment. j, HIF-1α cannot bind to most of its target genes in the HIFAL null condition. k Binding motifs enriched within 1 kb of the promoter region. l A dual heatmap illustrating the co-occupancy peaks of HIF1a and HIFAL ChIP-seq in MBA-MD-231 cells. Peaks were rank from the highest amount of HIF-1α/HIFAL to the lowest, and were flanking with ±0.5 kb sequence of TSS. Red indicates enrichment, while blue indicates no enrichment. m The enrichment peaks of HIFAL and HIF-1α overlap significantly. Under the most stringent criteria, 2804 out of the 3054 (91.8%) HIFAL peaks are also bound by HIF-1α. n Binding motifs enriched within ±0.5 kb of the promoter region. HIF-1α and HIFAL shares the most frequent target sequence. o Representative HIF-1α target glycolysis gene loci are similarly enriched by both HIF-1α and HIFAL in HIFAL WT or null MDA-MB-231 cells. Graphs show means ± SD of experimental triplicates, p values were determined by two-sided unpaired t-test (a–f, h). Source data are provided as a Source Data file. less

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The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.

In Nat Commun on 26 February 2021 by Zheng, F., Chen, J., et al.

Fig.1.A
Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
  • WB
  • Homo sapiens (Human)
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038... more
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038 at 2 h of HKII, LDHA, PDK1; p = 0.0219 at 4 h of GLUT1; p < 0.0001 at 8h-96h of GLUT1, HKII, LDHA, PDK1. b Hierarchical clustering for the lncRNAs differentially expressed in MDA-MB-231 and 76 N cells, cultured under normoxia (20%) or hypoxia (0.6%) conditions (fold change > 10, p < 0.05, two-sided paired t-test). c Microarray analysis of lncRNA expression in 4 pairs of breast cancer and adjacent normal tissues. (p < 0.05, two-sided paired t-test). d HIFAL level increases upon hypoxia, as measured by qRT-PCR. e, f HIFAL translocates into the nucleus upon hypoxia. The relative level of HIFAL in cytoplasmic and nuclear extract of MCF-7 (e) and MDA-MB-231 (f) cells were detected by qRT-PCR. Exogenous cel-miR-39 was added into the cytoplasmic or nuclear-fractionated RNA as an independent control. g–j After knocking down HIFAL, the kinetics of HIF-1α protein level under hypoxia was parallel with that of its target gene transcription. QRT-PCR was used to examine the mRNA expression of indicated genes GLUT1 (g), HKII (h), LDHA (i), PDK1 (j) in MDA-MB-231 cells (CTL vs LNA1 p = 0.015, p = 0.03, p = 0.04, p = 0.038, respectively, at 4 h in g, h, i, j. CTLvsLNA2 p = 0.02 at 4 h in g. CTLvsLNA1, p < 0.0001, p = 0.0012, p = 0.0062, p < 0.0001; CTLvs LNA2, p < 0.0001, p = 0.0011, p = 0.0159, p = 0.0002 at 8 h in g, h, i, j. CTLvsLNA1, CTLvsLNA2, p < 0.0001at 16h-96h in g, h, i, j. The HIFAL levels in cells are shown in the upper panel of (g). k, l Binding kinetics of HIF-1α to its target genes keeps increasing after hypoxia, which is dependent on HIFAL. The HIF-1α ChIP experiments were performed in the HIFAL KO (k) or WT MDA-MB-231 cells (l) at indicated time points after hypoxia and normalized by the corresponding HIF-1a protein levels. P = 0.0027, p = 0.0092, p = 0.0025, p = 0.0032 at 8 h, p < 0.0001 at 16h-64h of GLUT1, HKII, LDHA, PDK1. For a, d–l, p values were determined by two-sided unpaired t-test. Graphs show means ± SD of experimental triplicates. ***p < 0.001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file. less

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The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.

In Nat Commun on 26 February 2021 by Zheng, F., Chen, J., et al.

Fig.5.G
Fig.5.G showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
  • WB
  • Homo sapiens (Human)
HIFAL transcription is activated by HIF-1α transcriptional complex containing HIFAL itself in a feed-forward loop.a HIFAL transcription is not in line with HIF-1α protein level after hypoxia. b HIF-1α binds to the HIFAL promoter under hypoxia. MDA-MB-231 cells were cultured under hypoxia for 24 h... more
HIFAL transcription is activated by HIF-1α transcriptional complex containing HIFAL itself in a feed-forward loop.a HIFAL transcription is not in line with HIF-1α protein level after hypoxia. b HIF-1α binds to the HIFAL promoter under hypoxia. MDA-MB-231 cells were cultured under hypoxia for 24 h, then collected for ChIP assay. Primers spanning the HIFAL locus were used to confirm the binding specificity of HIF-1α at the promoter/HRE element. [HIFAL (B)], −916~−719bp [HIFAL (A)], and +578~+749 bp [(HIFAL(C)] of the promoter/HRE element. The β-actin promoter was used as the negative control. c Hypoxia-induced HIFAL transcription is inhibited by HIF-1α knockdown. MDA-MB-231 cells transiently transfected with siHIF-1α were collected to detect the HIFAL promoter transcription activity under hypoxia. d HIF-1α activates HIFAL transcription in a dose-dependent manner under hypoxia.HIF-1α plasmid (0, 0.25, 0.5, 1 μg) was transfected into MCF-7 cells for the detection of HIFAL transcriptional activity. e HRE mutation abolished the hypoxia induced HIFAL transcription activity. MDA-MB-231cells transfected with indicated wild type or mutant HIFAL promoter were collected to detect the promoter transcription activity. f PKM2 binds to the HIFAL promoter under hypoxia. MDA-MB-231 cells were collected for ChIP analyses. g Knocking down PKM2 reduces HIFAL level upon hypoxia, which could not be rescued by PKM1 overexpression. h PKM2 siRNA decreases the hypoxia-induced HIFAL promotor transcriptional activity, which could not be rescued by PKM1 overexpression. i PKM2 activates the HIFAL transcription in a dose-dependent manner under hypoxia. PKM2 plasmid (0, 0.25, 0.5, 1 μg) was transfected into MCF-7 cells for the detection of HIFAL transcriptional activity. j HIFAL binds to HIFAL promoter under hypoxia. MDA-MB-231 cells were transfected with biotinlabeled HIFAL or antisense, then collected for ChIP analyses. k, l Knocking down HIFAL decreases the hypoxia-induced HIFAL promotor transcriptional activity. The transcription activity of HIFAL promotor was shown in (k). The HIFAL levels are shown in (l). m HIFAL activates the transcriptional activity of HIFAL promotor in a dose-dependent manner under hypoxia. HIFAL plasmid (0, 0.25, 0.5, 1 μg) was used for the transfection into MCF-7 cells. For b, f, j, p < 0.0001 versus IgG precipitation. Graphs show means ± SD of experimental triplicates, p values were determined by two-sided unpaired t-test (a–m). Source data are provided as a Source Data file. less

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The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.

In Nat Commun on 26 February 2021 by Zheng, F., Chen, J., et al.

Fig.1.I
Fig.1.I showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
  • WB
  • Homo sapiens (Human)
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038... more
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038 at 2 h of HKII, LDHA, PDK1; p = 0.0219 at 4 h of GLUT1; p < 0.0001 at 8h-96h of GLUT1, HKII, LDHA, PDK1. b Hierarchical clustering for the lncRNAs differentially expressed in MDA-MB-231 and 76 N cells, cultured under normoxia (20%) or hypoxia (0.6%) conditions (fold change > 10, p < 0.05, two-sided paired t-test). c Microarray analysis of lncRNA expression in 4 pairs of breast cancer and adjacent normal tissues. (p < 0.05, two-sided paired t-test). d HIFAL level increases upon hypoxia, as measured by qRT-PCR. e, f HIFAL translocates into the nucleus upon hypoxia. The relative level of HIFAL in cytoplasmic and nuclear extract of MCF-7 (e) and MDA-MB-231 (f) cells were detected by qRT-PCR. Exogenous cel-miR-39 was added into the cytoplasmic or nuclear-fractionated RNA as an independent control. g–j After knocking down HIFAL, the kinetics of HIF-1α protein level under hypoxia was parallel with that of its target gene transcription. QRT-PCR was used to examine the mRNA expression of indicated genes GLUT1 (g), HKII (h), LDHA (i), PDK1 (j) in MDA-MB-231 cells (CTL vs LNA1 p = 0.015, p = 0.03, p = 0.04, p = 0.038, respectively, at 4 h in g, h, i, j. CTLvsLNA2 p = 0.02 at 4 h in g. CTLvsLNA1, p < 0.0001, p = 0.0012, p = 0.0062, p < 0.0001; CTLvs LNA2, p < 0.0001, p = 0.0011, p = 0.0159, p = 0.0002 at 8 h in g, h, i, j. CTLvsLNA1, CTLvsLNA2, p < 0.0001at 16h-96h in g, h, i, j. The HIFAL levels in cells are shown in the upper panel of (g). k, l Binding kinetics of HIF-1α to its target genes keeps increasing after hypoxia, which is dependent on HIFAL. The HIF-1α ChIP experiments were performed in the HIFAL KO (k) or WT MDA-MB-231 cells (l) at indicated time points after hypoxia and normalized by the corresponding HIF-1a protein levels. P = 0.0027, p = 0.0092, p = 0.0025, p = 0.0032 at 8 h, p < 0.0001 at 16h-64h of GLUT1, HKII, LDHA, PDK1. For a, d–l, p values were determined by two-sided unpaired t-test. Graphs show means ± SD of experimental triplicates. ***p < 0.001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file. less

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The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.

In Nat Commun on 26 February 2021 by Zheng, F., Chen, J., et al.

Fig.1.K
Fig.1.K showing Western Blotting in a Homo sapiens (Human) sample from the publication: The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis.
  • WB
  • Homo sapiens (Human)
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038... more
LncRNA HIFAL is essential for maintaining HIF-1α triggered transcription under hypoxia.a The kinetics of HIF-1α protein level under hypoxia, determined by immunoblotting, was not parallel with that of its target gene transcription by qRT-PCR in MDA-MB-231 cells. p = 0.0055, p = 0.0062, p = 0.0038 at 2 h of HKII, LDHA, PDK1; p = 0.0219 at 4 h of GLUT1; p < 0.0001 at 8h-96h of GLUT1, HKII, LDHA, PDK1. b Hierarchical clustering for the lncRNAs differentially expressed in MDA-MB-231 and 76 N cells, cultured under normoxia (20%) or hypoxia (0.6%) conditions (fold change > 10, p < 0.05, two-sided paired t-test). c Microarray analysis of lncRNA expression in 4 pairs of breast cancer and adjacent normal tissues. (p < 0.05, two-sided paired t-test). d HIFAL level increases upon hypoxia, as measured by qRT-PCR. e, f HIFAL translocates into the nucleus upon hypoxia. The relative level of HIFAL in cytoplasmic and nuclear extract of MCF-7 (e) and MDA-MB-231 (f) cells were detected by qRT-PCR. Exogenous cel-miR-39 was added into the cytoplasmic or nuclear-fractionated RNA as an independent control. g–j After knocking down HIFAL, the kinetics of HIF-1α protein level under hypoxia was parallel with that of its target gene transcription. QRT-PCR was used to examine the mRNA expression of indicated genes GLUT1 (g), HKII (h), LDHA (i), PDK1 (j) in MDA-MB-231 cells (CTL vs LNA1 p = 0.015, p = 0.03, p = 0.04, p = 0.038, respectively, at 4 h in g, h, i, j. CTLvsLNA2 p = 0.02 at 4 h in g. CTLvsLNA1, p < 0.0001, p = 0.0012, p = 0.0062, p < 0.0001; CTLvs LNA2, p < 0.0001, p = 0.0011, p = 0.0159, p = 0.0002 at 8 h in g, h, i, j. CTLvsLNA1, CTLvsLNA2, p < 0.0001at 16h-96h in g, h, i, j. The HIFAL levels in cells are shown in the upper panel of (g). k, l Binding kinetics of HIF-1α to its target genes keeps increasing after hypoxia, which is dependent on HIFAL. The HIF-1α ChIP experiments were performed in the HIFAL KO (k) or WT MDA-MB-231 cells (l) at indicated time points after hypoxia and normalized by the corresponding HIF-1a protein levels. P = 0.0027, p = 0.0092, p = 0.0025, p = 0.0032 at 8 h, p < 0.0001 at 16h-64h of GLUT1, HKII, LDHA, PDK1. For a, d–l, p values were determined by two-sided unpaired t-test. Graphs show means ± SD of experimental triplicates. ***p < 0.001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file. less

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p53 Is Regulated in a Biphasic Manner in Hypoxic Human Papillomavirus Type 16 (HPV16)-Positive Cervical Cancer Cells.

In Int J Mol Sci on 15 December 2020 by Zhuang, L., Ly, R., et al.

Fig.1.B
Fig.1.B showing Western Blotting from the publication: p53 Is Regulated in a Biphasic Manner in Hypoxic Human Papillomavirus Type 16 (HPV16)-Positive Cervical Cancer Cells.
  • WB
E6/E7 oncogene transcription and p53 protein expression in human papillomavirus (HPV)16-positive cervical cancer cells under normoxia and hypoxia. Cells were cultured for the indicated times at 21% O2 or at 1% O2. Total RNA and total protein were extracted. (A) qPCR analyses of HPV16 E6/E7 and va... more
E6/E7 oncogene transcription and p53 protein expression in human papillomavirus (HPV)16-positive cervical cancer cells under normoxia and hypoxia. Cells were cultured for the indicated times at 21% O2 or at 1% O2. Total RNA and total protein were extracted. (A) qPCR analyses of HPV16 E6/E7 and vascular endothelial growth factor (VEGF) transcripts. The 18S rRNA served as internal control for normalization. Error bars indicate the SD of three independent experiments. Corresponding transcript levels under normoxia were arbitrarily set to 1 and used as control for the calculation of significant differences (two-tailed Student’s t-test was employed, ** p < 0.01, *** p < 0.001). (B) Western blot analyses of cells after 24 h under normoxia/hypoxia. The filter was incubated with antibodies against p53 and hypoxia-inducible factor 1α (HIF-1α). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading and transfer control. (C) Time course of hypoxic incubation. Upper panels: qPCR analyses of HPV16 E6/E7 transcripts. The 18S rRNA served as internal control for normalization. Error bars indicate the SD of three independent experiments. The 0 h value of E6/E7 expression was set as a control for the calculation of significant differences (two-tailed Student’s t-test was employed, * p < 0.05, ** p < 0.01, *** p < 0.001 and ns, statistically not significant). Lower panels: Western blot analyses of p53 levels. GAPDH served as a loading control (S, short exposure; L, long exposure). The incubation time is indicated. The p53 ratio refers to normalized quantifications of p53 band intensities relative to GAPDH from three independent experiments (see Supplementary Table S1). less

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Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity.

In Int J Mol Sci on 10 December 2020 by Bouthelier, A., Meléndez-Rodríguez, F., et al.

Fig.1.B
Fig.1.B showing Western Blotting from the publication: Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity.
  • WB
Expression of hypoxia-inducible transcription factor (HIF)1α(P-A)2, HIF2α(P-A)2 and the HIF(P-A)2 (N2/C1), HIF(P-A)2 (N1/C2) chimeric versions in WT8 cells. (A) Scheme of the HIF1α(P-A)2 (in blue) and HIF2α(P-A)2 (in red), as well as the HIF1α/HIF2α chimeric constructs. The HIF(P-A)2 (N2/C1) cons... more
Expression of hypoxia-inducible transcription factor (HIF)1α(P-A)2, HIF2α(P-A)2 and the HIF(P-A)2 (N2/C1), HIF(P-A)2 (N1/C2) chimeric versions in WT8 cells. (A) Scheme of the HIF1α(P-A)2 (in blue) and HIF2α(P-A)2 (in red), as well as the HIF1α/HIF2α chimeric constructs. The HIF(P-A)2 (N2/C1) construct contains residues 1–414 of HIF2α, including the HIF2α basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) domain, and residues 412–826 of HIF1α that includes the HIF1α N-terminal transactivation domain/N-terminal transactivation domain (NTAD/CTAD) transactivation domains. The HIF(P-A)2 (N1/C2) construct contains amino acids 1–411 of HIF1α, including the HIF1α bHLH-PAS domain, and amino acids 415–870 of HIF2α that includes the HIF2α NTAD/CTAD transactivation domain; (B) representative Western blots probed for the HIF1α, HIF2α and β-actin proteins in control WT8 cells, and those expressing the HIF1α(P-A)2, HIF2α(P-A)2 and the HIF(P-A)2 (N2/C1) or HIF(P-A)2 (N1/C2) chimeric constructs. Circle indicates the HIF1α and HIF2α half where the antibodies against HIF1α or anti-HIF2α recognize. Estimated molecular weights of HIF1α and HIF2α (based on molecular weights markers included in the Western blot analysis) are included. less

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Signs of Chronic Hypoxia Suggest a Novel Pathophysiological Event in α-Synucleinopathies.

In Mov Disord on 1 December 2020 by Heras-Garvin, A., Danninger, C., et al.

Fig.1.B
Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Signs of Chronic Hypoxia Suggest a Novel Pathophysiological Event in α-Synucleinopathies.
  • WB
  • Homo sapiens (Human)
Signs of chronic hypoxia in MSA. (a) Schematic representation of the regulation of HIFα in normoxia and acute or chronic hypoxia. Genes coding for HIFα subunits are constitutively transcribed and translated. However, under normal oxygen conditions (normoxia), the resultant proteins are rapidly hy... more
Signs of chronic hypoxia in MSA. (a) Schematic representation of the regulation of HIFα in normoxia and acute or chronic hypoxia. Genes coding for HIFα subunits are constitutively transcribed and translated. However, under normal oxygen conditions (normoxia), the resultant proteins are rapidly hydroxylated, ubiquitinated, and degraded via proteasome in an oxygen‐dependent manner. Under acute hypoxic conditions, the decrease in oxygen levels impairs the hydroxylation, which results in HIF1α and HIF2α protein stabilization. Under chronic hypoxic conditions, HIF1α protein is degraded, and HIF2α drives the cellular adaptation. (b) Representative Western blot images showing HIF2α and HIF1α protein levels in extracts from human substantia nigra. Samples from control (c) and PD and MSA subjects were utilized. GAPDH, α‐tubulin, and β‐tubulin were used as loading controls. A protein extract from HEK cells exposed to DMOG, an agent that mimics hypoxic conditions by inhibiting PHDs, was used as a positive control (right lane). (c) Violin plot illustrating HIF2α protein levels in control and PD and MSA cases based on the quantification of Western blots. Controls, white circles; PD, black squares; MSA, green triangles; black triangles, PD cases that were clinically misdiagnosed as MSA. ANOVA, analysis of variance. ***P < 0.001, **P < 0.01 (Bonferroni's test). (d) Correlation analysis of HIF2α levels and disease duration in MSA and PD cases. MSA only: r2 = 0.1919, P = 0.069; PD only: r2 = 0.1211, P = 0.2677; MSA + PD: r2 = 0.1385, P = 0.0428. PD, black squares; MSA, green triangles; black triangles, PD cases that were clinically misdiagnosed as MSA. [Color figure can be viewed at wileyonlinelibrary.com] less

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Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.

In Mol Oncol on 1 June 2020 by Wu, J., Chai, H., et al.

Fig.5.A
Fig.5.A showing Western Blotting from the publication: Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.
  • WB
Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell ... more
Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell proliferation (mean ± SEM; n = 4; Student’s t‐test). **P < 0.01, ***P < 0.001, ****P < 0.0001. less

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Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.

In Mol Oncol on 1 June 2020 by Wu, J., Chai, H., et al.

Fig.5.C
Fig.5.C showing Western Blotting from the publication: Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer.
  • WB
Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell ... more
Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell proliferation (mean ± SEM; n = 4; Student’s t‐test). **P < 0.01, ***P < 0.001, ****P < 0.0001. less

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