Journal: Journal of Cellular and Molecular Medicine

Article Title: Neddylation inhibitor MLN4924 has anti‐HBV activity via modulating the ERK‐HNF1α‐C/EBPα‐HNF4α axis

doi: 10.1111/jcmm.16137

Figure Lengend Snippet: MLN4924 suppressed the expression of HNF1α, HNF4α and C/EBPα, required for HBV transcription. HepG2.2.15 cells were treated with 250 nM MLN4924, along with DMSO control for 48 h. Cells were then harvested for total RNA isolation or protein lysate preparation, followed by RT‐PCR analysis for indicated transcription factor (A‐D) and Western blotting with indicated Abs (E). Shown are mean ± SEM from three independent experiments ** P < .01, *** P < .001, NS, no significance

Article Snippet: The antibodies used were listed: anti‐p‐ERK (4370S, Cell Signaling Technology, USA), anti‐ERK (4695S, Cell Signaling Technology, USA), anti‐HNF1α (89670S,Cell Signaling Technology,USA), anti‐HNF4a(3113S, Cell Signaling Technology,USA), anti‐C/EBPa (8178S, Cell Signaling Technology, USA), anti‐PPARa (ab3484,abcam,USA), anti‐NEDD8 (ab81264, abcam, USA), anti‐GAPDH (2118S, Cell Signaling Technology, USA), anti‐Actin (A1015,DAWEN BIOTECH,CHINA) Normal Rabbit IgG (WD‐GAR007, DAWEN BIOTECH,CHINA),Normal Mouse IgG (GAM007,MULTI SCIENCES,CHINA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Neddylation inhibitor MLN4924 has anti‐HBV activity via modulating the ERK‐HNF1α‐C/EBPα‐HNF4α axis

doi: 10.1111/jcmm.16137

Figure Lengend Snippet: MLN4924 anti‐HBV model. MLN4924 has two distinct mechanisms of action. On one hand, MLN4924 inactivates CRLs to restore the levels of SMC5/6 to suppress cccDNA, ⁵⁰ and on the other hand, it activates ERK via EGFR signals to down‐regulate the expression of HNF1α, HNF4α and C/EBPα. Together, MLN4924 inhibits activities of various HBV promoters, leading to reduction of HBV RNA, HBsAg and rcDNA and finally virion production

Article Snippet: The antibodies used were listed: anti‐p‐ERK (4370S, Cell Signaling Technology, USA), anti‐ERK (4695S, Cell Signaling Technology, USA), anti‐HNF1α (89670S,Cell Signaling Technology,USA), anti‐HNF4a(3113S, Cell Signaling Technology,USA), anti‐C/EBPa (8178S, Cell Signaling Technology, USA), anti‐PPARa (ab3484,abcam,USA), anti‐NEDD8 (ab81264, abcam, USA), anti‐GAPDH (2118S, Cell Signaling Technology, USA), anti‐Actin (A1015,DAWEN BIOTECH,CHINA) Normal Rabbit IgG (WD‐GAR007, DAWEN BIOTECH,CHINA),Normal Mouse IgG (GAM007,MULTI SCIENCES,CHINA).

Techniques: Expressing

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

Article Title: Inflammation Induces Lipid Deposition in Kidneys by Downregulating Renal PCSK9 in Mice with Adriamycin-Induced Nephropathy

doi: 10.12659/MSM.917312

Figure Lengend Snippet: ( A–F ) Renal expression of TGF-β1, IL-1β, HNF1α, PCSK9, LDLR, and SREBP2 proteins (immunohistochemical analysis software was used to analyze the optical density values in the images, and semi-quantitative values of positive expression in kidney tissues were obtained) and mRNAs (from RT-qPCR, relative to GAPDH) in the 4 groups at 4, 8, and 12 weeks. Values represent means ± standard errors of mean for groups of 8 mice each. For comparisons at the same time point: * P <0.05 versus CTL, # P <0.05 versus AWI. TGF-β1 – transforming growth factor beta 1; IL-1β – interleukin 1β; HNF1α – hepatocyte nuclear factor 1α; PCSK9 – pro-protein convertase subtilisin kexin type 9; LDLR – low-density lipoprotein receptor; SREBP2 – sterol regulatory element binding protein 2; RT-q-PCR – real-time quantitative polymerase chain reaction; GAPDH – glyceraldehyde 3-phosphate dehydrogenase; CLT – control group; AWI group – Adriamycin-induced nephrosis with inflammation group.

Article Snippet: Immunohistochemistry (IHC) was used to examine the expression of interleukin (IL)-1β, transforming growth factor beta 1 (TGF-β1), HNF1α, PCSK9, LDLR, and SREBP2 using the following antibodies: rabbit anti-rat IL-1β antibody (working dilution 1: 100; sc-7884, Santa Cruz Biotechnology, USA), rabbit anti-rat TGF-β1 (working dilution 1: 100; sc-146, Santa Cruz Biotechnology), mouse anti-human SREBP2 antibody (working dilution 1: 100; sc-271615, Santa Cruz Biotechnology), mouse anti-human HNF1 α antibody (working dilution 1: 100; sc-393668, Santa Cruz Biotechnology,), rabbit anti-rat LDLR antibody (working dilution 1: 100; ab30532, Abcam, UK), rabbit anti-rat PCSK9 antibody (working dilution 1: 100; ab31762, Abcam).

Techniques: Expressing, Immunohistochemical staining, Software, Quantitative RT-PCR, Binding Assay, Real-time Polymerase Chain Reaction, Control

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

Article Title: Inflammation Induces Lipid Deposition in Kidneys by Downregulating Renal PCSK9 in Mice with Adriamycin-Induced Nephropathy

doi: 10.12659/MSM.917312

Figure Lengend Snippet: Representative immunostaining results of renal tissues for HNF1α, PCSK9, LDLR, and SREBP2 in each group at 4, 8, and 12 weeks. Scale bars indicate 100 μm. HNF1α – hepatocyte nuclear factor 1α; PCSK9 – pro-protein convertase subtilisin kexin type 9; LDLR – low-density lipoprotein receptor; SREBP2 – sterol regulatory element binding protein 2.

Article Snippet: Immunohistochemistry (IHC) was used to examine the expression of interleukin (IL)-1β, transforming growth factor beta 1 (TGF-β1), HNF1α, PCSK9, LDLR, and SREBP2 using the following antibodies: rabbit anti-rat IL-1β antibody (working dilution 1: 100; sc-7884, Santa Cruz Biotechnology, USA), rabbit anti-rat TGF-β1 (working dilution 1: 100; sc-146, Santa Cruz Biotechnology), mouse anti-human SREBP2 antibody (working dilution 1: 100; sc-271615, Santa Cruz Biotechnology), mouse anti-human HNF1 α antibody (working dilution 1: 100; sc-393668, Santa Cruz Biotechnology,), rabbit anti-rat LDLR antibody (working dilution 1: 100; ab30532, Abcam, UK), rabbit anti-rat PCSK9 antibody (working dilution 1: 100; ab31762, Abcam).

Techniques: Immunostaining, Binding Assay

Journal: Nature Communications

Article Title: Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus

doi: 10.1038/s41467-018-05782-5

Figure Lengend Snippet: IL-32γ downregulates HNF1α and HNF4α and reduces their binding to enhancers. a Map of liver-enriched transcription factor binding to HBV enhancers. b – d Huh7 cells were co-transfected with the HBV 1.2 and IL-32γ vectors or were treated with cytokines after HBV 1.2 transfection. The levels of transcription factors were determined by semi-quantitative RT-PCR ( b ), real-time PCR ( c ), or western blot analysis ( d . e , f ) Effect of IL-32γ and cytokines on the expression of HNF1α and HNF4α was analyzed by confocal fluorescence microscopy. At 24 h after transfection or treatment, immunofluorescence assay was performed using indicated antibodies (magnification, ×400; scale bar, 50 μm.). g , h Chromatin immunoprecipitation (ChIP) assay. Control or IL-32γ vector was transfected into Huh7 cells and ChIP assay was performed using anti-HNF4α ( g ) or anti-HNF1α ( h ) antibody. The level of the IL-32γ protein was determined by western blotting. The regions for R1–R3 were shown in above diagram ( a ). i Electrophoretic mobility shift assay (EMSA). An aliquot of 2 µg nuclear extracts was used. A cold competitor (50-fold) was used as a negative control. The protein complex was confirmed by western blotting using anti-HNF4α antibody. Data ( c ) was obtained from three independent experiments (mean ± S.D.). p < 0.001, p < 0.01 by Student’s t -test

Article Snippet: Primary antibodies against the following proteins and epitopes were used: HBV core protein (Dako, B0586, Hamburg, CA, USA, 1:2000), GFP (Sigma, G6795, 1:2000), HBsAg (Abcam, ab9193, Cambridge, UK, or Dako, 1:2000), HNF1α (Santa Cruz Biotechnology, sc-8986, 1:2000), HNF3β (Santa Cruz Biotechnology, sc-101060, 1:2000), CEBPα (Santa Cruz Biotechnology, sc-9314, 1:2000), HNF4α (Santa Cruz Biotechnology, sc-6556, 1:2000), Myc (Abcam, ab39688, 1:2000), lamin (Santa Cruz Biotechnology, sc-376248, 1:2000), NP (AbD Serotec, Raleigh, NC, USA, 1:2000), ICP27 (Abcam, 1:2000), tubulin (Santa Cruz Biotechnology, sc-8035, 1:2000), and actin (Sigma, A5316, 1:5000).

Techniques: Binding Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Fluorescence, Microscopy, Immunofluorescence, Chromatin Immunoprecipitation, Control, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Negative Control

Journal: Nature Communications

Article Title: Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus

doi: 10.1038/s41467-018-05782-5

Figure Lengend Snippet: Involvement of ERK1/2 signaling in IL-32γ-mediated inhibition of HBV replication. a Effect of IL-32γ and cytokines on MAPK signaling in Huh7 cells. TNF-α and IFN-γ were added for 30 min before harvest. Activation of MAPK signaling pathways was determined by western blotting with the indicated antibodies. b Effect of ERK1/2 activation inhibitor (10 µM U0126) on the expression of HNFs. c – e Effect of HNF4α ( c ), HNF1α ( d ), and U0126 ( e ) on IL-32γ-mediated inhibition of HBV replication. Viral replication and protein expression were determined by Southern and western blot analyses, respectively. U0126 was added for 48 h at a final concentration of 10 µM. Data ( c – e ) were obtained from three independent experiments (mean ± S.D.). p < 0.001, p < 0.05 by Student’s t -test

Article Snippet: Primary antibodies against the following proteins and epitopes were used: HBV core protein (Dako, B0586, Hamburg, CA, USA, 1:2000), GFP (Sigma, G6795, 1:2000), HBsAg (Abcam, ab9193, Cambridge, UK, or Dako, 1:2000), HNF1α (Santa Cruz Biotechnology, sc-8986, 1:2000), HNF3β (Santa Cruz Biotechnology, sc-101060, 1:2000), CEBPα (Santa Cruz Biotechnology, sc-9314, 1:2000), HNF4α (Santa Cruz Biotechnology, sc-6556, 1:2000), Myc (Abcam, ab39688, 1:2000), lamin (Santa Cruz Biotechnology, sc-376248, 1:2000), NP (AbD Serotec, Raleigh, NC, USA, 1:2000), ICP27 (Abcam, 1:2000), tubulin (Santa Cruz Biotechnology, sc-8035, 1:2000), and actin (Sigma, A5316, 1:5000).

Techniques: Inhibition, Activation Assay, Protein-Protein interactions, Western Blot, Expressing, Concentration Assay

Journal: Nature Communications

Article Title: Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus

doi: 10.1038/s41467-018-05782-5

Figure Lengend Snippet: IL-32γ is involved in cytokine-mediated suppression of HBV in PHHs. a The experimental scheme. PHHs were infected with 1000 genome equivalents HBV per cell. At 4 days post-infection (dpi), the HBeAg level was determined by ELISA. b Effect of IL-32 knockdown on cytokine-induced downregulation of HNF1α and HNF4α. PHHs were infected with HBV and shIL-32 lentivirus as described in a . Cytokines were added for 2 days before harvest. c , d Effect of IL-32 knockdown on cytokine-induced inhibition of HBV replication and HBeAg secretion. Aliquots of cell lysates were used for real-time PCR and HBeAg ELISA. e A hypothetical model of IL-32γ-mediated suppression of HBV through downregulation of HNF1α and HNF4α expression. When hepatocytes are infected with HBV, immune cells secrete TNF-α and IFN-γ, which induce the expression of IL-32γ. IL-32γ activates ERK1/2, which in turn downregulates the expression of HNF1α and HNF4α. Finally, the binding of HNFs to the viral enhancers/core promoter is reduced, which consequently inhibits HBV transcription and replication. Data ( c , d ) were obtained from three independent experiments (mean ± S.D.). p < 0.001 by Student’s t -test

Article Snippet: Primary antibodies against the following proteins and epitopes were used: HBV core protein (Dako, B0586, Hamburg, CA, USA, 1:2000), GFP (Sigma, G6795, 1:2000), HBsAg (Abcam, ab9193, Cambridge, UK, or Dako, 1:2000), HNF1α (Santa Cruz Biotechnology, sc-8986, 1:2000), HNF3β (Santa Cruz Biotechnology, sc-101060, 1:2000), CEBPα (Santa Cruz Biotechnology, sc-9314, 1:2000), HNF4α (Santa Cruz Biotechnology, sc-6556, 1:2000), Myc (Abcam, ab39688, 1:2000), lamin (Santa Cruz Biotechnology, sc-376248, 1:2000), NP (AbD Serotec, Raleigh, NC, USA, 1:2000), ICP27 (Abcam, 1:2000), tubulin (Santa Cruz Biotechnology, sc-8035, 1:2000), and actin (Sigma, A5316, 1:5000).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Knockdown, Inhibition, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Molecular and Cellular Biochemistry

Article Title: Up-regulation of PCSK9 gene expression and diminished level of LDL-receptor in rat liver as a potential cause of post-lipectomy hypercholesterolemia

doi: 10.1007/s11010-018-3484-8

Figure Lengend Snippet: The sequences of primers used in this study

Article Snippet: The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich).

Techniques: Sequencing

Journal: Molecular and Cellular Biochemistry

Article Title: Up-regulation of PCSK9 gene expression and diminished level of LDL-receptor in rat liver as a potential cause of post-lipectomy hypercholesterolemia

doi: 10.1007/s11010-018-3484-8

Figure Lengend Snippet: Analysis of HNF transcription factors in liver: a HNF1α mRNA relative levels; b HNF4α mRNA relative levels c representative western blot protein analysis of HNF1α and HNF4α standardized against actin of controls and lipectomized rats, d densitometric analysis of western blot HNF1α bands standardized against actin, e densitometric analysis of western blot HNF4α bands standardized against actin. a.u . arbitrary units. Data are presented as mean ± SD. * p < 0.05

Article Snippet: The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich).

Techniques: Western Blot

Journal: Molecular and Cellular Biochemistry

Article Title: Up-regulation of PCSK9 gene expression and diminished level of LDL-receptor in rat liver as a potential cause of post-lipectomy hypercholesterolemia

doi: 10.1007/s11010-018-3484-8

Figure Lengend Snippet: Subsequent inhibition of HNF1α and PCSK9 expression in HepG2 cells. silencing of HNF1α expression by two different siRNA ( a ) resulted in decrease of PSCK9 mRNA expression ( b ). Lipofectamine-treated cells were used as control. Graphs represent the mean ± SD of data from 6 cell plates in three separate experiments. a.u . arbitrary units.* p < 0.05

Article Snippet: The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich).

Techniques: Inhibition, Expressing

Journal: Molecular and Cellular Biochemistry

Article Title: Up-regulation of PCSK9 gene expression and diminished level of LDL-receptor in rat liver as a potential cause of post-lipectomy hypercholesterolemia

doi: 10.1007/s11010-018-3484-8

Figure Lengend Snippet: Proposed mechanism showing how HNFs may affect circulating LDL-cholesterol concentration in rats after lipectomy. Lipectomy causes increase of liver HNF4α and HNF1α. HNF1α activates Pcsk9 gene promoter leading to increased concentration of plasma PCSK9. PCSK9 binds to LDL-R and impairs proper scavenging of serum LDL-cholesterol. For details see “ ”

Article Snippet: The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich).

Techniques: Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: LTH-sEV promotes LSEC angiogenesis by regulating HNF1α/Ang-2 axis. ( A ). Diagram of a database cross to confirm transcription factor HNF1α; ( B ) qRT-PCR analysis of Ang-2 mRNA expression in LSECs transfected with pCtr or different doses of pHNF1α, n = 6; ( C ) Western blot analysis of HNF1α and Ang-2 protein expression in LSECs transfected with pCtr or different doses of pHNF1α, n = 3; ( D ) qRT-PCR analysis of Ang-2 mRNA expression in LSECs transfected with siCtr, siHNF1α-1or siHNF1α-2, n = 6; ( E ) Western blot analysis of HNF1α and Ang-2 protein expression in LSECs transfected with siCtr, siHNF1α-1, or siHNF1α-2, n = 3; ( F and G ) JASPAR database predicts the binding sites of HNF1α and Ang-2 and constructs the truncation; ( H ) Ang-2 promoter activity in LSECs transfected with different doses of pHNF1α or pCtr and pAng-2-2000; Ang-2 promoter activity in LSECs transfected with siCtr, siHNF1α-1or siHNF1α-2 and pAng-2-2000, n = 4; ( I ) Ang-2 promoter activity in 293T and LSECs transfected with pHNF1α and pAng-2, n = 4; ( J ) ChIP-PCR analysis of the binding area of HNF1α to Ang-2 in 293T and LSECs transfected with pCtr or pHNF1α, n = 4; ( K and L ) qRT-PCR and Western blot of HNF1α and Ang-2 protein expression in normal LSECs or LTH-sEV treated LSECs transfected with siCtr or siHNF1α, n = 6; ( M and N ) Fibrin gel bead assay and tube formation assay in LSECs or LTH-sEV-treated LSECs transfected with siCtr or siHNF1α, scale bar = 50/200 μm, n = 5; Compared with the siCtr group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Binding Assay, Construct, Activity Assay, Tube Formation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: LTH-sEV transports OGT into LSEC and liver to promote HNF1α O-GlcNAcylation. ( A ) qRT-PCR of HNF1α mRNA expression in LSECs treated with PBS, LTH-sEV (200, 400 μg/mL), n = 6; Compared with the NC group, ns, no significance; ( B ) IP-MS analysis of the protein binding to HNF1α in LSECs treated with PBS, LTH-sEV (400 μg/mL); ( C ) Co-IP of cell lysates from LSECs using antibodies against HNF1α; ( D ) Immunofluorescence images of the co-localization of OGT and HNF1α in LSECs, scale bar, 20 μm; ( E ) Western blot analysis of OGT expression in LTH-sEV; ( F ) qRT-PCR of OGT mRNA expression in LSECs treated with PBS, LTH-sEV (200, 400 μg/mL), n = 6; Compared with the PBS group, ns, no significance; ( G ) Western blot analysis of OGT, OGA, and O-GlcNac expression in LSECs treated with PBS, LTH-sEV (200, 400 μg/mL), n = 3; ( H ) Immunofluorescence images of co-localization of OGT and DiR-labeled LTH-sEV in LSEC, scale bar = 20 μm; ( I ) Western Blot analysis of OGT, OGA, and O-GlcNac protein expression in mouse liver. n = 3; ( J ) Immunofluorescence images of co-localization of OGT and CD63 in mouse liver, scale bar = 50 μm; ( K ) Immunohistochemistry images of OGT and O-GlcNac, scale bar = 50 μm, n = 6; ( L ) Co-IP of cell lysates from 293T transfected with V5-HNF1α using antibodies against V5-HNF1α; ( M ) Co-IP of cell lysates from LSECs using antibodies against HNF1α; ( N ) Co-IP of cell lysates from 293T transfected with V5-HNF1α and treated with PBS, LTH-sEV using antibodies against V5-HNF1α; ( O ) Co-IP of cell lysates from LSEC treated with PBS, LTH-sEV using antibodies against HNF1α.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Quantitative RT-PCR, Expressing, Protein-Protein interactions, Protein Binding, Co-Immunoprecipitation Assay, Immunofluorescence, Western Blot, Labeling, Immunohistochemistry, Transfection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: OGT promotes Ang-2 expression and LSEC angiogenesis through HNF1α O-GlcNAcylation at Ser471 site. ( A and B ) Co-IP of cell lysates from 293T transfected with V5-HNF1α and His-OGT or pCtr, siCtr, or siOGT using antibodies against V5-HNF1α; ( C ) Immunofluorescence images of HNF1α in LSEC transfected with shOGT or shCtr, scale bar = 10 μm; ( D ) YinOYang 1.2 website predicts O-GlcNAc sites of HNF1α; ( E ) Co-IP of cell lysates from 293T transfected with pCtr or various V5-HNF1α mutants using antibodies against V5-HNF1α; ( F ) Co-IP of cell lysates from 293T transfected with pCtr or various V5-HNF1α mutants and His-OGT using antibodies against V5-HNF1α; ( G ) qRT-PCR analysis of Ang-2 mRNA expression in 293T and LSECs transfected with pCtr and V5-HNF1α mutant; Compared with V5-HNF1α-WT group, ∗∗ P < .01; ∗∗∗ P < .001; ns, no significance; ( H ) Immunofluorescence images of V5 in LSEC transfected with pCtr, V5-HNF1α-WT or V5-HNF1α-S471A, scale bar = 10 μm; ( I–L ) Normal LSEC and OGT knocked down LSECs transfected with pCtr,V5-HNF1α-WT or V5-HNF1α-S471A; ( I ) qRT-PCR analysis of Ang-2 mRNA expression, n = 6; ( J ) Western blot analysis of Ang-2 protein expression, n = 3; ( K and L ) Fibrin gel bead assay and tube formation assay of LSEC, scale bar = 50/200 μm, n = 5. Compared with the pCtr group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ns, no significance.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Quantitative RT-PCR, Mutagenesis, Western Blot, Tube Formation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: OGT enriched MASLD-sEV promotes LSEC angiogenesis. ( A and B ) qRT-PCR and Western blot analysis of OGT expression in LSECs treated with Healthy-sEV and MASLD-sEV; ( C ) Immunofluorescence images of co-localization of OGT and DiR-labeled Healthy-sEV and MASLD-sEV, scale bar = 20 μm; ( D ) Co-IP of cell lysates from 293T transfected with V5-HNF1α and treated with Healthy-sEV and MASLD-sEV using antibodies against V5-HNF1α; ( E and F ) qRT-PCR and Western blot analysis of Ang-2 expression in LSEC treated with Healthy-sEV and MASLD-sEV, n = 6 or 3; ( G and H ) Fibrin gel bead assay and tube formation assay in LSECs treated with PBS, Healthy-sEV and MASLD-sEV, scale bar = 50/200 μm, n = 5; Compared with Healthy-sEV or NC group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ns, no significance.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Labeling, Co-Immunoprecipitation Assay, Transfection, Tube Formation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: BAGN inhibits LSEC capillarization and alleviates MASLD progression. ( A ) H&E staining of liver, heart, spleen, lung, and kidney in HFD mice treated with DMSO or BAGN (0.5 mg, 1.0 mg, 2.0 mg), scale bar = 50 μm; ( B ) Western blot analysis of O-GlcNac, OGT, and Ang-2 protein expression in HFD mice, n = 3; ( C ) Immunofluorescence images of HNF1α and O-GlcNac, scale bar = 20 μm; ( D ) SEM images and porosity of liver in each group, scale bar = 500 nm, n = 6; Immunohistochemistry images of CD31 and Ang-2, scale bar = 50 μm, n = 6; ( E ) Serum ALT, AST, and TG expression in each group, n = 6; ( F ) Serum IL-1β, IL-6, and TNFα expression in each group, n = 6; ( G ) Oil red O staining, immunohistochemistry images of α-SMA, Sirius red staining in each group, scale bar = 20/100/50 μm, n = 6; Compared with HFD group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Staining, Western Blot, Expressing, Immunofluorescence, Immunohistochemistry

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: HNF1 α inhibits the STAT3 pathway and promotes lipolytic catabolism lipid anabolism. (a) Western blot analysis showed that upregulation of HNF1 α expression promoted the expression of SOCS-3, inhibited the phosphorylation of STAT3, downregulated the expression of HNF1 α , inhibited the expression of SOCS-3, and promoted the phosphorylation of STAT3. (b) RT-qPCR and western blot showed that upregulation of HNF1 α expression inhibited the expression of SREBP-1c, promoted the expression of PPAR α , downregulated the expression of HNF1 α , promoted the expression of SREBP-1c, and inhibited the expression of PPAR α . Reported values are the means + SD of the three independent tests, with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: Western Blot, Expressing, Phospho-proteomics, Quantitative RT-PCR

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: STAT3 inhibitor NSC74859 can reverse the effect of the downregulation of HNF1 α expression on hepatic glycolipid metabolism. (a) Oil Red O staining test showed that NSC74859 reduced the number of red granular lipid droplets in LO2 cells induced by FFA. Downregulation of HNF1 α expression followed by addition of NSC74859 reduced FAR-induced red granule lipid droplets in LO2 cells. (b) Biochemical indicators showed that NSC74859 reduced TC, TG, and NEFA contents. After downregulating the HNF1 α expression and adding NSC74859, the contents of TC, TG, and NEFA decreased significantly. (c, d) Western blot analysis showed that NSC74859 promoted IRS-1 and AKT phosphorylation. Downregulation of HNF1 α expression followed by addition of NSC74859 abolished the inhibition of IRS-1 and AKT phosphorylation by downregulating the HNF1 α expression. (e) Western blot analysis showed that NSC74859 inhibited the expression of SREBP-1c and promoted the expression of PPAR α . Downregulation of HNF1 α expression followed by NSC74859 reversed the effects of downregulation of HNF1 α expression on SREBP-1c and PPAR α expression. Reported values are the means + SD of the three independent tests, with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: Expressing, Staining, Western Blot, Phospho-proteomics, Inhibition

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: HNF1 α inhibits steatosis through suppressing STAT3 in vivo . (a) Oil Red O staining showed that increased liver steatosis in HNF1 α -/- mouse liver compared with WT HFD group. Treatment of NSC74859 in HNF1 α -/- mice rescued them from severe steatosis. (b) Liver weight of HNF1 α -/- mice were significantly higher than WT mice fed with HFD, treatment of NSC74859 decreased mouse liver weight. (c–e) Biochemical indicators showed that HNF1 α deficiency increased triglyceride (TG), cholesterol (TC), and nonesterified fatty acid (NEFA) contents from the liver tissue. After treating the mice with NSC74859, the contents of TG, TC, and NEFA decreased significantly. (f, g) Serum fasting insulin levels were determined by ELISA, and homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as [FBG (mmol/l) × FIns (mIU/l)]/22.5. n = 4–8 per group, at the 8th week. HNF1 α defect mice were significantly higher than WT mice fed with HFD. After treating the mice with NSC74859, the insulin levels and the HOMA-IR index decreased significantly. (h) Western blot analysis showed that HNF1 α deficiency increased the expression of SREBP-1c and phosphorylation of STAT3 and reduced the expressions of SOCS3 and PPAR α and phosphorylation of IRS-1 and AKT. Treating HNF1 α defect mice with NSC74859 reversed these protein expressions: NSC74859 inhibited the expression of SREBP-1c and promoted the expression of PPAR α . All values are expressed as mean ± SEM, n = 8–12 per group, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Phospho-proteomics

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: HNF1 α inhibits the STAT3 pathway and promotes lipolytic catabolism lipid anabolism. (a) Western blot analysis showed that upregulation of HNF1 α expression promoted the expression of SOCS-3, inhibited the phosphorylation of STAT3, downregulated the expression of HNF1 α , inhibited the expression of SOCS-3, and promoted the phosphorylation of STAT3. (b) RT-qPCR and western blot showed that upregulation of HNF1 α expression inhibited the expression of SREBP-1c, promoted the expression of PPAR α , downregulated the expression of HNF1 α , promoted the expression of SREBP-1c, and inhibited the expression of PPAR α . Reported values are the means + SD of the three independent tests, with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: Western Blot, Expressing, Phospho-proteomics, Quantitative RT-PCR

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: STAT3 inhibitor NSC74859 can reverse the effect of the downregulation of HNF1 α expression on hepatic glycolipid metabolism. (a) Oil Red O staining test showed that NSC74859 reduced the number of red granular lipid droplets in LO2 cells induced by FFA. Downregulation of HNF1 α expression followed by addition of NSC74859 reduced FAR-induced red granule lipid droplets in LO2 cells. (b) Biochemical indicators showed that NSC74859 reduced TC, TG, and NEFA contents. After downregulating the HNF1 α expression and adding NSC74859, the contents of TC, TG, and NEFA decreased significantly. (c, d) Western blot analysis showed that NSC74859 promoted IRS-1 and AKT phosphorylation. Downregulation of HNF1 α expression followed by addition of NSC74859 abolished the inhibition of IRS-1 and AKT phosphorylation by downregulating the HNF1 α expression. (e) Western blot analysis showed that NSC74859 inhibited the expression of SREBP-1c and promoted the expression of PPAR α . Downregulation of HNF1 α expression followed by NSC74859 reversed the effects of downregulation of HNF1 α expression on SREBP-1c and PPAR α expression. Reported values are the means + SD of the three independent tests, with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: Expressing, Staining, Western Blot, Phospho-proteomics, Inhibition

Journal: Journal of Diabetes Research

Article Title: HNF1 α Controls Liver Lipid Metabolism and Insulin Resistance via Negatively Regulating the SOCS-3-STAT3 Signaling Pathway

doi: 10.1155/2019/5483946

Figure Lengend Snippet: HNF1 α inhibits steatosis through suppressing STAT3 in vivo . (a) Oil Red O staining showed that increased liver steatosis in HNF1 α -/- mouse liver compared with WT HFD group. Treatment of NSC74859 in HNF1 α -/- mice rescued them from severe steatosis. (b) Liver weight of HNF1 α -/- mice were significantly higher than WT mice fed with HFD, treatment of NSC74859 decreased mouse liver weight. (c–e) Biochemical indicators showed that HNF1 α deficiency increased triglyceride (TG), cholesterol (TC), and nonesterified fatty acid (NEFA) contents from the liver tissue. After treating the mice with NSC74859, the contents of TG, TC, and NEFA decreased significantly. (f, g) Serum fasting insulin levels were determined by ELISA, and homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as [FBG (mmol/l) × FIns (mIU/l)]/22.5. n = 4–8 per group, at the 8th week. HNF1 α defect mice were significantly higher than WT mice fed with HFD. After treating the mice with NSC74859, the insulin levels and the HOMA-IR index decreased significantly. (h) Western blot analysis showed that HNF1 α deficiency increased the expression of SREBP-1c and phosphorylation of STAT3 and reduced the expressions of SOCS3 and PPAR α and phosphorylation of IRS-1 and AKT. Treating HNF1 α defect mice with NSC74859 reversed these protein expressions: NSC74859 inhibited the expression of SREBP-1c and promoted the expression of PPAR α . All values are expressed as mean ± SEM, n = 8–12 per group, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The antibodies used included HNF1 α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPAR α antibody (Abcam, ab8934).

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Phospho-proteomics