Journal: Frontiers in Immunology

Article Title: In vitro study: HIF-1α-dependent glycolysis enhances NETosis in hypoxic conditions

doi: 10.3389/fimmu.2025.1583587

Figure Lengend Snippet: Time-dependent effect of hypoxia on NETosis formation. The upper part shows the expression of HIF-1α and citH3 in dHL−60 cells incubated under hypoxic conditions for various times (0–4 h) with or without PMA, assessed by Western blot analysis, with β-actin and H3 as loading controls. The lower part shows the quantification of HIF-1α and citH3 protein levels normalized to β-actin or H3, respectively. n = 6. ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The workstation was purged with gas, sealed, and placed in a conventional incubator at 37°C for 4 h. To inhibit HIF-1α expression, 10 μM LW6 (HY-13671, MedChemExpress, USA) was added to the dHL-60 cells following the initial 30-minute incubation.

Techniques: Expressing, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: In vitro study: HIF-1α-dependent glycolysis enhances NETosis in hypoxic conditions

doi: 10.3389/fimmu.2025.1583587

Figure Lengend Snippet: LW6 inhibits HIF-1α expression and reverses hypoxia-induced NETosis. The upper part shows the expression of HIF-1α and citH3 in differentiated dHL−60 cells incubated under hypoxic conditions for 4 h with or without LW6, assessed by Western blot analysis, with β-actin and H3 as loading controls. The lower part shows the quantification of HIF-1α and citH3 protein levels normalized to β-actin or H3, respectively. n = 3 biological replicates, with three technical replicates per sample. ****p < 0.0001, ns, not significant.

Article Snippet: The workstation was purged with gas, sealed, and placed in a conventional incubator at 37°C for 4 h. To inhibit HIF-1α expression, 10 μM LW6 (HY-13671, MedChemExpress, USA) was added to the dHL-60 cells following the initial 30-minute incubation.

Techniques: Expressing, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: In vitro study: HIF-1α-dependent glycolysis enhances NETosis in hypoxic conditions

doi: 10.3389/fimmu.2025.1583587

Figure Lengend Snippet: HIF-1α plays a key regulatory role in hypoxia-induced glycolysis and NETosis. dHL−60 were incubated under hypoxic conditions for various times (4 h) with or without LW6 (A) or Bay-876 (B) , and the expression of GLUT-1, HK2, PFK1, GAPDH, ENO1, PKM2, LDHA, and citH3 were detected by western blot. Expression of glycolysis‐related enzymes and citH3 protein levels were normalized to the β–actin or H3 protein, respectively. n = 3 biological replicates, with three technical replicates per sample. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: The workstation was purged with gas, sealed, and placed in a conventional incubator at 37°C for 4 h. To inhibit HIF-1α expression, 10 μM LW6 (HY-13671, MedChemExpress, USA) was added to the dHL-60 cells following the initial 30-minute incubation.

Techniques: Incubation, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: In vitro study: HIF-1α-dependent glycolysis enhances NETosis in hypoxic conditions

doi: 10.3389/fimmu.2025.1583587

Figure Lengend Snippet: HIF-1α-dependent glycolysis regulates NETosis during hypoxia. (A) Representative fluorescence micrographs depicting NET-release of neutrophils. (B) Quantification of the mean fluorescence intensity (MFI) of each group of SYTOX Green. (C) Percentage of NETosis. n=6. ***p < 0.001, ****p < 0.0001.

Article Snippet: The workstation was purged with gas, sealed, and placed in a conventional incubator at 37°C for 4 h. To inhibit HIF-1α expression, 10 μM LW6 (HY-13671, MedChemExpress, USA) was added to the dHL-60 cells following the initial 30-minute incubation.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: In vitro study: HIF-1α-dependent glycolysis enhances NETosis in hypoxic conditions

doi: 10.3389/fimmu.2025.1583587

Figure Lengend Snippet: Graphical Abstract. Under hypoxic conditions, HIF-1α is activated in the neutrophil nucleus. This activation promotes the transcription of key glycolytic enzymes, including GLUT1, HK2, PFK1, GAPDH, ENO1, PKM2, and LDHA, thereby enhancing intracellular glycolysis and increasing lactate production. Elevated lactate may activate PADI4, which in turn induces histone citrullination and chromatin decondensation, leading to the release of NETs into the extracellular space (left panel). Treatment with HIF-1α inhibitor LW6 or glycolysis inhibitor Bay-876 blocks this pathway, effectively reversing NETosis under hypoxic conditions (right panel), highlighting the critical role of HIF-1α-dependent glycolysis in NETosis under low oxygen stress.

Article Snippet: The workstation was purged with gas, sealed, and placed in a conventional incubator at 37°C for 4 h. To inhibit HIF-1α expression, 10 μM LW6 (HY-13671, MedChemExpress, USA) was added to the dHL-60 cells following the initial 30-minute incubation.

Techniques: Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Long non-coding RNA and mRNA profile analysis of metformin to reverse the pulmonary hypertension vascular remodeling induced by monocrotaline.

doi: 10.1016/j.biopha.2019.108933

Figure Lengend Snippet: Fig. 4. Signal pathway identification and transcription factor activity verification. A: Representative western blotting strips of PAs from MCT-induced PAH rats with or without metformin treatment and densitometric analysis showing Hif1α, PKG relative to β-actin; B: Representative western blotting strips of PAs from MCT- induced PAH rats with or without metformin treatment and densitometric analysis showing p-AKT relative to AKT; C: Representative western blotting strips of PAs from MCT-induced PAH rats with or without metformin treatment and densitometric analysis showing p53 relative to β-actin; D: Representative western blotting strips of PAs from MCT-induced PAH rats with or without metformin treatment and densitometric analysis showing Sp1, Est1 relative to β-actin. MCT means monocrotaline; Met means metformin. **p < 0.01. n = 3.

Article Snippet: The dilution ratio of all primary antibodies used was as follows: (PKG, catalog number: 21646-1-AP, 1:1000 dilution, proteintech), (Sp1, catalog number: 21962-1-AP, 1:2000 dilution, proteintech), (Est1, catalog number: 12522-1-AP, 1:1000 dilution, proteintech), (Hif1α, Cat. #: AF1009, 1:2000 dilution, Affinity Biosciences), (p53, catalog number: 10442-1-AP, 1:5000 dilution, proteintech), (Cyclin D1, catalog number: 26755-1-AP, 1:1000 dilution, proteintech), (β-actin, catalog number: 60008-1-1 g, 1:10000 dilution, proteintech), (AKT, catalog number: 10176-2-AP, 1:2000 dilution, proteintech), (p-AKT, # 9271, 1:1000 dilution, Cell Signaling Technology).

Techniques: Activity Assay, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Long non-coding RNA and mRNA profile analysis of metformin to reverse the pulmonary hypertension vascular remodeling induced by monocrotaline.

doi: 10.1016/j.biopha.2019.108933

Figure Lengend Snippet: Fig. 7. Mechanism analysis of NONRATT015587.2 regulation of pulmonary vascular remodeling in vitro assay and differential gene clustergram in MCT-induced PAH rats and controls, or metformin treatment. A-G: Realtime-PCR analysis of p53, Est1, Rela, Cebpa/b, Sp1, Hif1α expression in pcDNA, pcDNA- NONRATT015587.2, and pcDNA- NONRATT015587.2+metformin group. H: Representative western blotting strips of PASMCs from pcDNA-NONRATT015587.2 with or without metformin treatment and densitometric analysis showing Hif1α, p53 relative to β-actin; I: The differential expression of mRNA in the microarray analysis. Hierarchical clustering analysis is shown for 32 mRNA (fold change > 2, p < 0.05) in the control, monocrotaline and monocrotaline + metformin group using gene microarray analysis. MCT means monocrotaline; Met means metformin. **p < 0.01. n = 3.

Article Snippet: The dilution ratio of all primary antibodies used was as follows: (PKG, catalog number: 21646-1-AP, 1:1000 dilution, proteintech), (Sp1, catalog number: 21962-1-AP, 1:2000 dilution, proteintech), (Est1, catalog number: 12522-1-AP, 1:1000 dilution, proteintech), (Hif1α, Cat. #: AF1009, 1:2000 dilution, Affinity Biosciences), (p53, catalog number: 10442-1-AP, 1:5000 dilution, proteintech), (Cyclin D1, catalog number: 26755-1-AP, 1:1000 dilution, proteintech), (β-actin, catalog number: 60008-1-1 g, 1:10000 dilution, proteintech), (AKT, catalog number: 10176-2-AP, 1:2000 dilution, proteintech), (p-AKT, # 9271, 1:1000 dilution, Cell Signaling Technology).

Techniques: In Vitro, Expressing, Western Blot, Quantitative Proteomics, Microarray, Control

Journal: iScience

Article Title: Long-term intermittent hypoxia in mice induces inflammatory pathways implicated in sleep apnea and steatohepatitis in humans

doi: 10.1016/j.isci.2024.108837

Figure Lengend Snippet:

Article Snippet: Antibodies used for Western blots are as follows: anti-HIF1a (Abcam, ab2185), anti-HIF1a (Santa Cruz Biotechnologies, sc13515), anti-α-TUBULIN (Sigma, T5168), anti-Rabbit HRP (Jackson laboratories, 111-035-003), anti-Mouse HRP (Jackson laboratories, 115-035-003).

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Reverse Transcription, Avidin-Biotin Assay, Gene Expression, Software, Gas Chromatography-Mass Spectrometry, Microscopy, Luminex