recombinant mouse netrin 4  (R&D Systems)

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Netrin‐1 is a HSC‐enriched secreted factor in the liver. (A) Heatmap of HSC‐enriched genes encoding secreted factors in the liver ( GSE129516 ). (B) UMAP plots showing relative Ntn‐1 mRNA expression across liver cell types. (C) Violin plots of Ntn‐1 gene expression. (D) Immunoblotting of isolated mouse primary hepatocytes (MPH), primary HSCs (pHSCs), and NPCs with pHSCs depleted (NPC * ). (E,F) Immunofluorescence staining for Netrin‐1, α‐SMA, and F4/80 in mouse liver. Representative images. Scale bar: 100 µm.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Expressing, Gene Expression, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Induction of Netrin‐1 expression in MASH and fibrotic liver. Mice were fed a chow (n = 3) or GAN (n = 3) diet for 5 months. (A, B) Plasma ALT and AST levels. (C) H&E (top) and Sirius Red (bottom) staining of liver sections (scale bar = 100 µm). (D–F) qPCR analysis of hepatic gene expression. (G) Immunoblotting of total liver lysate. (H‐N) Mice received i.p. injections of vehicle or CCl4 (0.6 ml/kg body weight) twice weekly for 3 weeks and were sacrificed 4 days after the final injection (n = 5). (H, I) Plasma ALT and AST levels. (J) H&E (top) and Sirius Red (bottom) staining of liver sections (scale bar = 100 µm). (K, L) qPCR analysis of hepatic gene expression. (M) qPCR analysis of Nnt1 expression. (N) Immunoblotting of total liver lysate. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Expressing, Clinical Proteomics, Staining, Gene Expression, Western Blot, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: AAV‐mediated overexpression of Netrin‐1 exacerbates liver fibrosis. AAV8‐TBG‐Vector (AAV‐Vector) and AAV8–TBG‐Ntn1(AAV‐Ntn1) were administered prior to GAN diet feeding. The following parameters were measured in AAV‐vector (n = 5) and AAV‐Ntn1 mice (n = 5) fed a GAN diet for 4 months: (A–C) Body weight, liver weight, and liver‐to‐body weight ratio in transduced mice. (D, E) Plasma ALT and AST levels. (F) H&E (top), Sirius Red (middle), and Masson's trichrome staining (bottom) of liver sections (scale bar = 100 µm). (G) Quantification of Sirius Red‐positive areas in liver sections. (H) Liver hydroxyproline content. (I) qPCR analysis of hepatic gene expression. (J) Immunoblotting of total liver lysate. (K) Heatmap of differentially expressed genes in the liver. (L) Gene ontology analysis of upregulated genes in AAV‐Ntn1 mice. (M) Correlation of differentially expressed genes in diet‐induced MASH livers ( GSE129516 ) versus Ntn1 overexpression. (N–P) Heatmaps of hepatocyte‐, HSC‐, and macrophage‐enriched genes regulated by Ntn1 overexpression. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Over Expression, Plasmid Preparation, Clinical Proteomics, Staining, Gene Expression, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: HSC‐specific Netrin‐1 deletion alleviates liver fibrosis following GAN diet feeding.Ntn1 fl/fl (n = 7) and Ntn1 fl/fl Lrat‐Cre (n = 8) mice were fed a GAN diet for 5 months. (A) qPCR analysis of hepatic Ntn1 expression. (B–F) Body and tissue weight. (G, H) Plasma ALT and AST levels. (I) H&E (top), Sirius Red (middle), and Masson trichrome staining (bottom) of liver sections (scale bar = 100 µm). (J) Quantification of Sirius Red‐positive areas in liver sections. (K) Liver hydroxyproline content. (L, M) qPCR analysis of hepatic gene expression. (N) Immunoblotting of total liver lysate. (O) Heat map of differentially expressed genes. (P) Gene ontology analysis of downregulated gene clusters. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Expressing, Clinical Proteomics, Staining, Gene Expression, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Netrin‐1 promotes fibrotic gene expression by stimulating calcium signaling in HSCs. (A) Volcano plot of differentially expressed genes in mHSCs treated with rhNetrin‐1 protein or PBS for 12 hrs. (B) Gene set enrichment analysis of upregulated genes. (C) qPCR analysis of fibrotic gene expression. (D) Immunofluorescence staining of α‐SMA (red) and Vimentin (green) in mHSCs treated with PBS or rhNetrin‐1 (300 ng/mL) for 24 hrs. Nuclei, DAPI (blue). (E) Wound‐healing assay of mHSCs after rhNetrin‐1 treatment; migration rate quantified on the right. (F) Immunoblotting of total cell lysates 24 hrs post‐treatment. (G) Calcium imaging traces in mHSCs treated with PBS (n = 6), Netrin‐1 (300ng/ml, n = 6), angiotensin II (10ng/ml, n = 9). Arrows indicate treatment initiation. Data are presented as mean ± SD. (H) Gene expression analysis of mHSCs pretreated with or without 10µM KN93, followed by PBS or Netrin‐1 treatment. (I) Immunoblotting of total cell lysates. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Gene Expression, Immunofluorescence, Staining, Wound Healing Assay, Migration, Western Blot, Imaging, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: UBC5B is required for HSC activation in response to Netrin‐1. (A) Violin plots showing Unc5b gene expression in each liver cell cluster ( GSE129516 ). (B) qPCR analysis of Netrin‐1 receptor gene expression in mHSCs. (C) Immunoblotting of isolated pHSC, MPH, and NPC*. (D–F) mHSCs were transduced with lentivirus‐carrying vectors containing the indicated shRNAs or an empty vector (shCtrl). (D) qPCR analysis of Unc5b expression. (E) Calcium imaging traces in mHSCs treated with phosphate buffer saline or Netrin‐1. (F) qPCR analysis of fibrosis‐related gene expression. (G) Immunoblotting of total cell lysates. (H–P) Eight‐week‐old C57BL/6 mice were injected with 1 x 10^8 TU/mouse lentivirus particles via the tail vein, followed by 2‐month HFMCD feeding (n = 4 per group). (H,I) Body and liver weight. (J, K) Serum ALT and AST levels. (L) Relative Unc5b expression in lung, kidney, and liver tissues. (M) H&E (top), Sirius Red (middle), and Masson trichrome staining (bottom) of liver sections (scale bar = 100 µm). (N) Quantification of Sirius Red‐positive areas in liver sections and hydroxyproline content in liver tissue. (O) qPCR analysis of hepatic fibrosis‐ and inflammation‐related genes in liver tissues. (P) Immunoblotting of total liver lysate. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Activation Assay, Gene Expression, Western Blot, Isolation, Transduction, Plasmid Preparation, Expressing, Imaging, Saline, Injection, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: LNP‐mediated RNAi knockdown of Netrin‐1 mitigates liver fibrosis. (A) Schematic diagram of LNP production and experimental design. (B, C) qPCR analysis of gene expression in mHSCs treated with vehicle or siNtn1‐LNP for 24 hrs. (D) Immunoblotting of total cell lysates. (E–M) Mice were fed with HFMCD for 12 weeks and administered with Ctrl‐LNP (n = 6) or siNtn1‐LNP (n=6) via tail vein twice a week for 2 weeks. (E, F) Body and liver weight of treated mice. (G, H) Plasma ALT and AST level. (I) H&E (top), Sirius Red (middle), and Masson's trichrome staining (bottom) of liver sections (scale bar = 100 µm). (J) Quantification of Sirius Red‐positive area in liver sections. (K) Hydroxyproline content in liver tissue. (L) qPCR analysis of hepatic gene expressions. (M) Immunoblotting of total liver lysates. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, ***p < 0.001, two‐tailed unpaired Student's t ‐test.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Knockdown, Gene Expression, Western Blot, Clinical Proteomics, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Autocrine Netrin‐1 signaling promotes HSC activation and liver fibrosis. Netrin‐1 expression is upregulated in HSCs during metabolic dysfunction–associated steatohepatitis and injury‐mediated liver fibrosis. Secreted Netrin‐1 establishes an autocrine positive feedback loop by binding to UNC5B receptors on HSCs. Receptor activation triggers calcium influx and profibrotic response in HSCs, leading to increased ECM production and liver fibrosis.

Article Snippet: The mHSCs were treated with 300 ng/ml recombinant Netrin‐1 protein (R&D System, 1109‐N1‐025) or 10 ng/ml ang II (Selleck, P1085) in the presence of an indicator.

Techniques: Activation Assay, Expressing, Binding Assay

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Netrin‐1 is a HSC‐enriched secreted factor in the liver. (A) Heatmap of HSC‐enriched genes encoding secreted factors in the liver ( GSE129516 ). (B) UMAP plots showing relative Ntn‐1 mRNA expression across liver cell types. (C) Violin plots of Ntn‐1 gene expression. (D) Immunoblotting of isolated mouse primary hepatocytes (MPH), primary HSCs (pHSCs), and NPCs with pHSCs depleted (NPC * ). (E,F) Immunofluorescence staining for Netrin‐1, α‐SMA, and F4/80 in mouse liver. Representative images. Scale bar: 100 µm.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Expressing, Gene Expression, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Induction of Netrin‐1 expression in MASH and fibrotic liver. Mice were fed a chow (n = 3) or GAN (n = 3) diet for 5 months. (A, B) Plasma ALT and AST levels. (C) H&E (top) and Sirius Red (bottom) staining of liver sections (scale bar = 100 µm). (D–F) qPCR analysis of hepatic gene expression. (G) Immunoblotting of total liver lysate. (H‐N) Mice received i.p. injections of vehicle or CCl4 (0.6 ml/kg body weight) twice weekly for 3 weeks and were sacrificed 4 days after the final injection (n = 5). (H, I) Plasma ALT and AST levels. (J) H&E (top) and Sirius Red (bottom) staining of liver sections (scale bar = 100 µm). (K, L) qPCR analysis of hepatic gene expression. (M) qPCR analysis of Nnt1 expression. (N) Immunoblotting of total liver lysate. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Expressing, Clinical Proteomics, Staining, Gene Expression, Western Blot, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: AAV‐mediated overexpression of Netrin‐1 exacerbates liver fibrosis. AAV8‐TBG‐Vector (AAV‐Vector) and AAV8–TBG‐Ntn1(AAV‐Ntn1) were administered prior to GAN diet feeding. The following parameters were measured in AAV‐vector (n = 5) and AAV‐Ntn1 mice (n = 5) fed a GAN diet for 4 months: (A–C) Body weight, liver weight, and liver‐to‐body weight ratio in transduced mice. (D, E) Plasma ALT and AST levels. (F) H&E (top), Sirius Red (middle), and Masson's trichrome staining (bottom) of liver sections (scale bar = 100 µm). (G) Quantification of Sirius Red‐positive areas in liver sections. (H) Liver hydroxyproline content. (I) qPCR analysis of hepatic gene expression. (J) Immunoblotting of total liver lysate. (K) Heatmap of differentially expressed genes in the liver. (L) Gene ontology analysis of upregulated genes in AAV‐Ntn1 mice. (M) Correlation of differentially expressed genes in diet‐induced MASH livers ( GSE129516 ) versus Ntn1 overexpression. (N–P) Heatmaps of hepatocyte‐, HSC‐, and macrophage‐enriched genes regulated by Ntn1 overexpression. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Over Expression, Plasmid Preparation, Clinical Proteomics, Staining, Gene Expression, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: HSC‐specific Netrin‐1 deletion alleviates liver fibrosis following GAN diet feeding.Ntn1 fl/fl (n = 7) and Ntn1 fl/fl Lrat‐Cre (n = 8) mice were fed a GAN diet for 5 months. (A) qPCR analysis of hepatic Ntn1 expression. (B–F) Body and tissue weight. (G, H) Plasma ALT and AST levels. (I) H&E (top), Sirius Red (middle), and Masson trichrome staining (bottom) of liver sections (scale bar = 100 µm). (J) Quantification of Sirius Red‐positive areas in liver sections. (K) Liver hydroxyproline content. (L, M) qPCR analysis of hepatic gene expression. (N) Immunoblotting of total liver lysate. (O) Heat map of differentially expressed genes. (P) Gene ontology analysis of downregulated gene clusters. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Expressing, Clinical Proteomics, Staining, Gene Expression, Western Blot, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Netrin‐1 promotes fibrotic gene expression by stimulating calcium signaling in HSCs. (A) Volcano plot of differentially expressed genes in mHSCs treated with rhNetrin‐1 protein or PBS for 12 hrs. (B) Gene set enrichment analysis of upregulated genes. (C) qPCR analysis of fibrotic gene expression. (D) Immunofluorescence staining of α‐SMA (red) and Vimentin (green) in mHSCs treated with PBS or rhNetrin‐1 (300 ng/mL) for 24 hrs. Nuclei, DAPI (blue). (E) Wound‐healing assay of mHSCs after rhNetrin‐1 treatment; migration rate quantified on the right. (F) Immunoblotting of total cell lysates 24 hrs post‐treatment. (G) Calcium imaging traces in mHSCs treated with PBS (n = 6), Netrin‐1 (300ng/ml, n = 6), angiotensin II (10ng/ml, n = 9). Arrows indicate treatment initiation. Data are presented as mean ± SD. (H) Gene expression analysis of mHSCs pretreated with or without 10µM KN93, followed by PBS or Netrin‐1 treatment. (I) Immunoblotting of total cell lysates. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Gene Expression, Immunofluorescence, Staining, Wound Healing Assay, Migration, Western Blot, Imaging, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: UBC5B is required for HSC activation in response to Netrin‐1. (A) Violin plots showing Unc5b gene expression in each liver cell cluster ( GSE129516 ). (B) qPCR analysis of Netrin‐1 receptor gene expression in mHSCs. (C) Immunoblotting of isolated pHSC, MPH, and NPC*. (D–F) mHSCs were transduced with lentivirus‐carrying vectors containing the indicated shRNAs or an empty vector (shCtrl). (D) qPCR analysis of Unc5b expression. (E) Calcium imaging traces in mHSCs treated with phosphate buffer saline or Netrin‐1. (F) qPCR analysis of fibrosis‐related gene expression. (G) Immunoblotting of total cell lysates. (H–P) Eight‐week‐old C57BL/6 mice were injected with 1 x 10^8 TU/mouse lentivirus particles via the tail vein, followed by 2‐month HFMCD feeding (n = 4 per group). (H,I) Body and liver weight. (J, K) Serum ALT and AST levels. (L) Relative Unc5b expression in lung, kidney, and liver tissues. (M) H&E (top), Sirius Red (middle), and Masson trichrome staining (bottom) of liver sections (scale bar = 100 µm). (N) Quantification of Sirius Red‐positive areas in liver sections and hydroxyproline content in liver tissue. (O) qPCR analysis of hepatic fibrosis‐ and inflammation‐related genes in liver tissues. (P) Immunoblotting of total liver lysate. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01; two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Activation Assay, Gene Expression, Western Blot, Isolation, Transduction, Plasmid Preparation, Expressing, Imaging, Saline, Injection, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: LNP‐mediated RNAi knockdown of Netrin‐1 mitigates liver fibrosis. (A) Schematic diagram of LNP production and experimental design. (B, C) qPCR analysis of gene expression in mHSCs treated with vehicle or siNtn1‐LNP for 24 hrs. (D) Immunoblotting of total cell lysates. (E–M) Mice were fed with HFMCD for 12 weeks and administered with Ctrl‐LNP (n = 6) or siNtn1‐LNP (n=6) via tail vein twice a week for 2 weeks. (E, F) Body and liver weight of treated mice. (G, H) Plasma ALT and AST level. (I) H&E (top), Sirius Red (middle), and Masson's trichrome staining (bottom) of liver sections (scale bar = 100 µm). (J) Quantification of Sirius Red‐positive area in liver sections. (K) Hydroxyproline content in liver tissue. (L) qPCR analysis of hepatic gene expressions. (M) Immunoblotting of total liver lysates. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, ***p < 0.001, two‐tailed unpaired Student's t ‐test.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Knockdown, Gene Expression, Western Blot, Clinical Proteomics, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine Netrin‐1 Signaling in Hepatic Stellate Cells Drives Liver Fibrosis and Diet‐Induced Metabolic Dysfunction‐Associated Steatohepatitis in Mice

doi: 10.1002/advs.202514545

Figure Lengend Snippet: Autocrine Netrin‐1 signaling promotes HSC activation and liver fibrosis. Netrin‐1 expression is upregulated in HSCs during metabolic dysfunction–associated steatohepatitis and injury‐mediated liver fibrosis. Secreted Netrin‐1 establishes an autocrine positive feedback loop by binding to UNC5B receptors on HSCs. Receptor activation triggers calcium influx and profibrotic response in HSCs, leading to increased ECM production and liver fibrosis.

Article Snippet: Prior to treatments, cells were switched to serum‐free DMEM for 12 h. Treatments included Netrin‐1 (R&D System, 1109‐N1‐025) and KN93 (MCE, HY‐15465).

Techniques: Activation Assay, Expressing, Binding Assay

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: (A) Experimental scheme showing the transduction of Adenovirus-Cre or -empty to pancreatic organoids isolated from LSL-Kras G12D/+ mice. d, day. Adeno, adenovirus. Scale bar, 20 μm. (B) qRT-PCR for axon guidance molecules and neurotrophins using the pancreatic organoids. n = 7 mice. A.U., arbitrary units. Two-tailed unpaired Student’s t-tests. (C) qRT-PCR for Ntn1 using pancreatic tissues from wild-type (WT), Pdx1-Cre; LSL-Kras G12D/+ (KC), and Pdx1-Cre; LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ (KPC) mice. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (D) Co-immunofluorescence (Co-IF) for NTN1 and CK19 (a duct marker) and quantification of the ratio of NTN1 + cells in CK19 + ductal cells. Normal pancreas from WT mice and pancreatic intraepithelial neoplasia (PanIN1, 2, and 3) and pancreatic ductal adenocarcinoma (PDAC) areas from KPC mice were evaluated. The inset shows a magnified view of normal ductal cells lacking NTN1 + signals. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (E) qRT-PCR for NTN1 receptors using pancreatic tissues from WT, KC, and KPC mice. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (F) Co-immunofluorescence for NEO1 and CK19 and quantification of the ratio of NEO1 + cells in CK19 + ductal cells. Normal pancreas from WT mice and PanIN and PDAC areas from KPC mice were evaluated. The inset shows a magnified view of normal ductal cells lacking NEO1 + signals. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (G) Co-immunofluorescence for NTN1 and CK19 using human normal pancreas and PDAC tissues. CK19 + ductal areas are magnified in the insets. n = 12 patients with PDAC. Two-tailed unpaired Student’s t-test. Scale bars, 50 μm. (H) The expression of NTN1 and NEO1 in bulk RNA-sequencing data using human pancreatic tissues. n = 165 (normal) and 178 patients (PDAC). The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets were analyzed. FPKM, fragments per kilobase of transcript per million mapped reads. Two-tailed unpaired Student’s t-tests. In all figures, bar graphs show mean ± s.e.m (standard error of the mean), and asterisks denote the following P-values. ****, P-value < 0.0001; ***, P-value of 0.0001 to 0.001; **, P-value of 0.001 to 0.01; *, P-value of 0.01 to 0.05; ns, P-value ≥ 0.05. A , Created in BioRender. Wu, F. (2025) https://BioRender.com/mlrkrl8

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Transduction, Isolation, Quantitative RT-PCR, Two Tailed Test, Comparison, Immunofluorescence, Marker, Expressing, RNA Sequencing

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: (A) qRT-PCR for Ntn1 and Neo1 using murine LSL-Kras G12D/+ pancreatic organoids transduced with Adenovirus-Cre or -Empty. See Fig. 1A for the experimental schematic. 24-hour treatment with the MEK inhibitor trametinib (10 nM) or vehicle was performed 6 days after adenovirus transduction. n = 6 each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (B) Co-immunofluorescence for NTN1, phosphorylated ERK (pERK), and CK19. Normal pancreas from WT mice and pancreatic intraepithelial neoplasia (PanIN1, 2, and 3) from KC mice were evaluated. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Statistical analysis was performed using the percentage of NTN1 + pERK + cells in total CK19 + cells (red). n = 4 mice each. Scale bars, 50 μm. (C) Experimental scheme showing the isolation of pancreatic organoids from KC mice and treatment of the KC organoids with vehicle, isoproterenol (ISO; β agonist; 0.1 μM), ICI118,551 (ICI; β2-specific antagonist; 10 μM), and trametinib (10 nM) for 6 days. Scale bar, 20 μm. (D) qRT-PCR for Ntn1 and Neo1 using KC pancreatic organoids treated with the indicated drugs. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (E) Experimental scheme showing in vivo treatment of KC mice (16-20 weeks of age) with vehicle, ISO (10 μg/g/day), and ISO + ICI (0.2 μg/g/day). These drugs were intraperitoneally injected into KC mice daily for 6 days. (F) Co-immunofluorescence for NTN1 (upper panel) and NEO1 (lower panel) with CK19 using pancreatic tissues from KC mice treated with vehicle, ISO, or ISO+ICI. n = 5 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 50 μm. C and E , Created in BioRender. Wu, F. (2025) https://BioRender.com/mlrkrl8

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Mutagenesis, Quantitative RT-PCR, Transduction, Comparison, Immunofluorescence, Isolation, In Vivo, Injection

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: (A and B) Celiac Ganglia (CG) were isolated from Th-Cre/Rosa26-tdtomato mice, and the CG tissue explants were treated with recombinant NTN1 (rNTN1; 100 ng/mL) and NEO1-blocking antibody (NEO1 Ab; 500 ng/mL) for 7 days. (A) Experimental scheme. (B) Representative images and quantification of tdtomato + neurite outgrowth. White dotted lines indicate the areas of neurite elongation. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (C) Experimental schematic showing epithelial knockout of Ntn1 in the KC mouse model. M, months. (D) Percentage of PanIN lesions in the total pancreatic tissue area was evaluated using hematoxylin and eosin (H&E)-stained sections from the pancreas of Ntn1 -WT KC and Ntn1 -cKO (conditional KO) KC mice. Green areas denote PanIN lesions. n = 5 mice each. KCN, Pdx1-Cre; LSL-Kras G12D ; Ntn1 flox/flox . Two-tailed unpaired Student’s t-tests. Scale bars, 1 mm. (E) Whole-mount staining for tyrosine hydroxylase (TH) was performed using optically cleared pancreatic tissue from WT, KC, and KCN mice at 12 months of age. Autofluorescence was used to visualize the pancreatic structure. n = 4 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 1 mm. (F ) Experimental scheme showing the injection of the retrograde neuronal tracer Fast Blue into the pancreas of WT, KC, and KCN mice. (G) Fast Blue (FB) fluorescence and TH immunofluorescence signals were evaluated using CGs from WT, KC, and KCN mice 1 week after FB injection. Areas surrounded by yellow dotted lines indicate the CG tissues. Red arrowheads denote neurons double-positive for TH and FB. n = 4 mice each. See Supplementary Fig. S3J for images from the individual channels (TH and FB). One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 50 μm. A, C, and F , Created in BioRender. Wu, F. (2025) https://BioRender.com/mlrkrl8

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Isolation, Recombinant, Blocking Assay, Comparison, Knock-Out, Staining, Two Tailed Test, Injection, Fluorescence, Immunofluorescence

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: (A) KC organoids (pancreatic organoids isolated from Pdx1-Cre; LSL-Kras G12D/+ mice) were treated with recombinant NTN1 (rNTN1; 100 ng/mL) and NEO1-blocking antibody (NEO1 Ab; 500 ng/mL) for 6 days. The number and area of organoids were evaluated. n = 3 mice each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (B) qRT-PCR for Zeb1 and Vim using sg-control- and sg- Neo1 -expressing mT4 cells treated with rNTN1 (100 ng/mL) for 48 hours. n = 3 each. sg, single guide RNA. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. ( C) Western blotting for ZEB1, VIM, and SOX9 using sg-ctrl (control) and sg- Neo1 mT4 cells treated with rNTN1 (100 ng/mL) for 48 hours. ( D) Sphere assay using sg-ctrl and sg- Neo1 mT4 cells treated with rNTN1 (100 ng/mL) for 6 days. Arrowheads denote cancer spheres. n = 5 each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (E) qRT-PCR for Sox9 , Sox2, Cd44, and Prom1 using sg-ctrl and sg- Neo1 mT4 cells treated with rNTN1 (100 ng/mL) for 48 hours. n = 4 each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (F) mT4 organoids were treated with rNTN1 (100 ng/mL), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL), and FAK inhibitor (FAKi; defactinib; 1 μM) for 6 days. n = 4 each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. Scale bars, 100 μm. (G) qRT-PCR for Zeb1, Vim, and Sox9 using mT4 organoids treated with rNTN1 (100 ng/mL), NEO1 Ab (500 ng/mL), and FAKi (defactinib; 1 μM) for 48 hours. n = 4 each. One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests. (H) Kaplan-Meier survival curves. n = 16 (KPC) and 15 mice (KPCN). w, weeks. Log-rank test. (I) Co-immunofluorescence for phosphorylated FAK (pFAK) and CK19 using pancreatic tumors from KPC and KPCN mice. n = 6 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 50 μm. (J-K) Co-immunofluorescence for ZEB1 (J) and SOX9 (K) with CK19 using pancreatic tumors from KPC and KPCN mice. The nuclear ZEB1 + or SOX9 + area in CK19 + cells was quantified. n = 4 mice each. Two-tailed unpaired Student’s t-tests. Scale bars, 50 μm.

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Activation Assay, Isolation, Recombinant, Blocking Assay, Comparison, Quantitative RT-PCR, Control, Expressing, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: (A) Experimental schematic showing a PDAC liver metastasis model generated by splenic injection of NTN1-overexpressing (NTN1-OE) and control mT4 cells. w, weeks. (B) Kaplan-Meier survival curves. n = 8 mice each. Log-rank test. (C) Histological tumor areas were evaluated using livers collected 2 weeks after splenic injection. Green areas denote tumor regions. n = 5 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 2 mm. (D) Whole-mount staining for PGP9.5 was performed using optically cleared liver tissues. n = 4 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 2 mm. (E) Immunofluorescence for TH using mT4 liver metastasis. Red arrowheads denote TH + adrenergic nerves. n = 4 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 50 μm. (F and G) Co-immunofluorescence for ZEB1 (F) and SOX9 (G) with CK19 using mT4 liver metastasis. The nuclear ZEB1 + or SOX9 + area in CK19 + cells was quantified. n = 5 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 50 μm. A , Created in BioRender. Wu, F. (2025) https://BioRender.com/mlrkrl8

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Generated, Injection, Control, Two Tailed Test, Staining, Immunofluorescence

Journal: Cancer research

Article Title: Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1

doi: 10.1158/0008-5472.CAN-25-2243

Figure Lengend Snippet: Blockade of Ntn1 or Neo1 inhibits the features of EMT and cancer stemness, and restrains the progression of PDAC liver metastasis. (A) Experimental scheme showing a liver metastasis model generated by splenic injection of control (sg-control) and Neo1 -knockout mT4 cells which express sg Neo1 #1. w, weeks. (B) Kaplan-Meier survival curves. n = 10 mice each. Log-rank test. (C) Histological assessment of tumor area was performed 2 weeks after splenic injection. Green areas denote tumor areas. n = 5 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 2 mm. (D and E) Co-immunofluorescence for ZEB1 (D) and SOX9 (E) with CK19 using mT4 liver metastasis. The nuclear ZEB1 + or SOX9 + area in CK19 + cells was quantified. n = 5 mice each. Two-tailed unpaired Student’s t-tests. Scale bars, 100 μm. (F) Experimental scheme showing a liver metastasis model generated by splenic injection of mT4 cells and treatment of tumor-bearing mice with a NTN1-neutralizing antibody (NTN1 Ab; NP137) or isotype antibody. Mice were intraperitoneally injected with NTN1 Ab (10 mg/kg) or isotype IgG antibody every other day. (G) Kaplan-Meier survival curves. n = 7 mice each. Log-rank test. (H) Histological assessment of tumor areas was performed 2 weeks after splenic injection. Green areas denote tumor areas. n = 7 mice each. Two-tailed unpaired Student’s t-test. Scale bars, 2 mm. (I and J) Co-immunofluorescence for ZEB1 (I) and SOX9 (J) with CK19 using mT4 liver metastasis. The nuclear ZEB1 + or SOX9 + area in CK19 + cells was quantified. n = 5 mice each. Two-tailed unpaired Student’s t-tests. Scale bars, 100 μm. A and F , Created in BioRender. Wu, F. (2025) https://BioRender.com/mlrkrl8

Article Snippet: After 24 hours, the cells were treated with recombinant NTN1 (rNTN1; 100 ng/mL; 1109-N1; R&D Systems), NEO1-blocking antibody (NEO1 Ab; 500 ng/mL; AF1079; R&D Systems; RRID: AB_2151002), and FAK inhibitor (defactinib; 1 μM; HY-12289A; MedChem Express) for 6 days in advanced DMEM containing 0.5% fetal bovine serum (FBS) (Gemini).

Techniques: Generated, Injection, Control, Knock-Out, Two Tailed Test, Immunofluorescence