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

Journal: Redox Biology

Article Title: Porcine epidemic diarrhea virus promotes viral replication via ROS/HIF-1α-mediated glycolysis

doi: 10.1016/j.redox.2026.104008

Figure Lengend Snippet: PEDV infection activates HIF-1α signaling to promote viral replication. (A) KEGG pathway enrichment analysis of differential metabolites was identified by targeted metabolomics. The top 20 enriched pathways were shown based on P-values. (B–C) The expression of HIF-1α and PEDV N protein in mock- and PEDV-infected ST cells were determined by western blots. β-Tubulin was used as a control. (D) ST cells were treated with the HIF-1α activator DMOG (50 μM) or the HIF-1α inhibitor KC7F2 (40 μM), respectively, for 24 h, followed by PEDV infection for another 24 h. Viral RNA levels of PEDV N were quantified by qRT-PCR. (E–G) Protein levels of HIF-1α and PEDV N were determined by western blots in cells treated as described in (D). Data were presented as mean ± SEM from three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: KC7F2 (HIF-1α inhibitor, HY-18777), dimethyloxalylglycine (DMOG, HIF-1α activator, HY-15893), N -Acetyl- l -cysteine (NAC; HY-B0215), Rotenone (ROT; HY-B1756), and Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; HY-100941) were purchased from MedChemExpress (MCE, China). (L)-Sodium lactate (Lac; T4103) was purchased from TargetMol (China).

Techniques: Infection, Expressing, Western Blot, Control, Quantitative RT-PCR

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: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: Wild-type C57BL/6 mice were fed an MCD or MCS diet for 8 weeks. (A) Liver sections were stained with H&E or Oil-Red-O to assess liver injury and steatosis. (B) RNA isolated from either total liver or liver mononuclear cells (LMNC) was used to measure HIF-1α expression by qPCR. (C) The protein levels of HIF-1α, BNIP3, Beclin-1, LC3-II and p62 in total liver were assessed by western blot, quantified using ImageJ analysis and normalized to β-actin. Mice experiments are representative of 3–10 mice per experimental group. *P<0.05.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Staining, Isolation, Expressing, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: Liver sections from MCD or MCS diet-fed mice were subjected to F4/80 immunohistochemistry (black arrows indicate the F4/80 positive cells), the F4/80 RNA levels were measured by qPCR in corresponding liver samples. (B) The protein levels of HIF-1α, BNIP3, Beclin-1, LC3-II and p62 were assessed in isolated hepatic macrophages by western blot. Image density was quantified using ImageJ analysis and normalized to β-actin. Mice experiments are representative of 3–10 mice per experimental group. *P<0.05.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Immunohistochemistry, Isolation, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: Liver tissues and blood samples were collected from either NASH patients or healthy controls. (A) Liver sections were subjected to F4/80 immunohistochemistry (black arrows indicate the F4/80 positive cells). The total number of F4/80-positive cells from five high-powered fields was counted per liver section by microscopy. At least three liver sections were included in each group. (B) Circulating monocytes were isolated from blood samples, the protein levels of HIF-1α, BNIP3, Beclin-1, LC3 I, LC3 II and P62 in monocytes were assessed by western blot, quantified using ImageJ analysis and normalized to beta-actin. Representative blots are shown from 10 healthy donors and 12 patients with diagnosed NASH. *P<0.05. Cont, control.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Immunohistochemistry, Microscopy, Isolation, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: LysMCreHIFdPAfl/fl mice were generated by cross-breeding LysMCre mice, which express Cre recombinase in myeloid cells under the control of the lysozyme M promoter, with HIFdPAfl/fl mice, which lacks proline hydroxylation sites for HIF degradation. (A) Genotyping analysis of the HIFdPAfl/fl and LysMCreHIFdPAfl/fl mice. (B) The mRNA levels of HIF-1α in BMDMs, hepatic macrophages, and hepatocytes from HIFdPAfl/fl and LysMCreHIFdPAfl/fl mice were measured by qRT-PCR. (C) BNIP3 and p62 mRNA levels in BMDMs and MCP-1, IL-1β, and IL-10 mRNA expression in liver macrophages were measured by qRT-PCR. LC3-II and p62 protein levels were measured by western blot, quantified using ImageJ analysis, and normalized to β-actin. (D) Liver sections were stained with H&E or Oil-Red-O to assess liver injury and steatosis. (E) ALT levels were assessed from serum samples. (F) mRNA levels of MCP-1, TNF-α and PAI-1 were measured in the total liver by qRT-PCR. Mice experiments are representative of 3–10 mice per experimental group. *P<0.05.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Generated, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: (A) Bone marrow from WT mice was isolated, and BMDMs generated after 7 days in the presence of L929 cell supernatant. Cells were stimulated with 250 μM PA for different time points and HIF-1α, p62, and LC3-II were assessed by Western blot. (B) BMDMs were cultivated in control medium or under PA stimulation for 6 or 12 hours with or without bafilomycin (Baf, 100 nM) (added at 0h or for the last 2 hours for 6 and 12 h incubations). (C) Human monocytes from healthy volunteers were isolated and differentiated to macrophages and HIF-1α, p62, and LC3-II were assessed in PA-stimulated cells by Western blot, quantified using ImageJ analysis and normalized to β-actin. (D) LC3-II levels of human macrophages were assessed in the presence or absence of bafilomycin (100nM) (added as in B) in PA-stimulated cells. Representative blots are shown from at least three independent experiments. *P<0.05. C, control.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Isolation, Generated, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: THP-1 cells were differentiated by 100nM PMA for 48h and treated with either 250 μM PA for the indicated time points, or BSA as a control. (A) The mRNA expression of HIF-1α and its target genes BNIP3, PAI-1 and Glut-1 were measured by qPCR. (B) Nuclear HIF-1α protein expression was measured via western blot and quantified using ImageJ analysis. (C) HIF-1α DNA-binding activity was tested and quantified using the electrophoretic mobility shift assay. (D) The detection of LC3 puncta in THP-1 cells was performed with a rabbit anti-LC3 antibody and immunofluorescence staining. THP-1 cells with more than 10 punctate dots were considered as positive cells (right), and the number and size of LC3 puncta/cell were quantified (left). Quantification of LC3-positive THP-1 cells and representative blots were performed in three independent experiments. (E) Protein levels of BNIP3 and Beclin-1 were assessed by western blot, quantified using ImageJ analysis, and normalized to β-actin. (F) Cells were cultivated in control medium or under PA stimulation for 12 hours with or without bafilomycin (Baf, 100 nM) for the last 2 hours. LC3 II and p62 protein levels were measured by western blot and quantified using ImageJ analysis. *P<0.05. Cont., control.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Expressing, Western Blot, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Immunofluorescence, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: HIF-1α mRNA and protein levels were downregulated using HIF-1α siRNA (A) or overexpressed using the HIF-1α-bearing pcDNA3 expression vector (B) in THP-1 macrophages. HIF-1α mRNA levels were assessed by qPCR. Protein levels of HIF-1α, BNIP3, Beclin-1, and LC3-II were assessed at indicated time points by western blot after transfection, quantified using ImageJ analysis and normalized to β-actin. HIF-1α siRNA (C) or overexpressed (D) THP-1 macrophages were cultivated in control medium or under PA stimulation with or without bafilomycin (100 nM) for the last 2 hours. LC3-II and P62 protein levels were measured by western blot, and the LC3-II ratio was calculated.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Expressing, Plasmid Preparation, Western Blot, Transfection

Journal: Hepatology (Baltimore, Md.)

Article Title: Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis

doi: 10.1002/hep.30215

Figure Lengend Snippet: (A) NF-κB DNA-binding activity was measured after PA stimulation at indicated time points using EMSA. TNF-α, IL-1β and IL-6 levels in the supernatants of THP-1 cells upon PA treatment were measured using an ELISA assay. (B) Phosphorylated p65 (P-p65) levels in THP-1 macrophages treated with HIF-1α siRNA were measured by western blot and quantified using ImageJ analysis. (C) Cells were pretreated for 30 min with an autophagy inducer, rapamycin (Rap, 100 nM) or inhibitor, 3-methyladenine (3-MA, 5 mM) prior to 6h-PA stimulation. Protein levels of P-p65 and P65 were measured by western blot and quantified using ImageJ analysis. (D) Cells were transfected using control or ATG5 siRNA, and P-p65 levels were measured by western blot and quantified using ImageJ analysis. (E) Cells were cultivated in control medium or under PA stimulation for 6 hours with or without bafilomycin (Baf, 100 nM) (added at 0h or for the last 2 hours for 6 and 12 h incubations), and P-p65 levels were measured by western blot. (F) TNF-α, IL-1β and IL-6 levels in the supernatants were measured by ELISA. Representative blots and ELISAs are shown from at least three independent experiments *P<0.05.

Article Snippet: Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay was performed on equal amounts (5–10 μg) of nuclear protein extracts from THP-1 macrophages using 50,000 cpm of γ- 32 P-labeled HIF-1α consensus oligonucleotide as we previously described (Santa Cruz, CA, USA) ( 14 ).

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection