Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: Nogo-B is hyperexpressed in fibrotic liver macrophages and is positively correlated with the degree of fibrosis. ( A ) Normal and fibrotic liver sections from patients were subjected to H&E and Sirius Red staining and Masson staining and α-SMA IHC analysis (original magnification 1×; scale bar = 2000 μm). ( B ) Nogo-B mRNA expression in human liver tissues was quantified via RT-PCR. ( C ) Nogo-B protein expression in human normal and fibrotic liver tissues was explored via Western blotting with respect to the loading control GAPDH. ( D ) Nogo-B mRNA expression in different types of human fibrotic liver cells was identified via qRT-PCR. ( E ) Nogo-B protein expression in different types of human fibrotic liver cells was explored via Western blotting with respect to the loading control GAPDH. ( F–G ) Dual IF staining of Nogo-B ( green ) and CD68 ( red ) in human liver tissues, and the ratio of double-positive cells per high-power field was calculated (original magnification 20×; scale bar = 100 μm). ( H–I ) Western blotting was used to measure the ER-stress markers XBP1s, ATF6, ATF4, and CHOP in normal and fibrotic human liver, and GAPDH served as the loading control. ( J ) Measurement of Nogo-B in serum of patients with hepatic fibrosis were measured by ELISA. ( K ) Nogo-B mRNA expression in murine livers analyzed by qRT-PCR. ( L ) Nogo-B protein levels in murine liver macrophages determined by Western blotting with respect to the loading control GAPDH. ( M–N ) Dual-IF staining of Nogo-B ( red ) and F4/80 ( green ) in murine livers, with quantification of double-positive cells per high-power field (n = 6/group; original magnification 20×; scale bar = 100 μm). ( O ) Multiple IF analysis revealed colocalization of CD68 ( purple ), Nogo-B ( green ), SOX9 ( red ), and α-SMA ( yellow ) in normal and fibrotic liver tissues (original magnification 20×; scale bar = 100 μm). ( P ) Multiplex IF revealed colocalization of F4/80 ( purple ), Nogo-B ( green ), SOX9 ( red ), and α-SMA ( yellow ) in livers from BDL mice and in control samples. (original magnification 10×; scale bar = 200 μm). Human samples: control (n = 18), mild fibrosis (n = 20), advanced fibrosis (n = 13); mice samples (n = 6 per group). The data are presented as the means ± SEMs; ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Multiplex Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: Myeloid-specific Nogo-B deficiency alleviates liver injury and fibrosis. ( A ) Breeding scheme used to generate mice with myeloid-specific Nogo-B deletion. ( B ) Identification of myeloid-specific Nogo-B-deficient mice. ( C ) Serum Nogo-B concentrations in the 3 liver fibrosis models, as determined by ELISA. ( D ) Measurement of ALT and AST in the serum of the mice. ( E ) Collagen I and α-SMA mRNA expression in the murine livers was evaluated through qRT-PCR. ( F–H ) Liver sections from the mice were subjected to H&E and Sirius Red staining and Masson staining and α-SMA IHC analysis, and the proportions of Sirius red-positive and Masson-positive and a-SMA-positive regions were quantified. ( I–K ) Hepatic protein levels of collagen I, MMP-9, α-SMA, and TIMP-1 were assessed via Western blotting; GAPDH served as the loading control. The data are presented as the means ± SEMs; n = 6 per group; original magnification, 10×; scale bars, 200 μm; ∗∗ P < .01.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Staining, Western Blot, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: Myeloid Nogo-B deficiency suppresses inflammation and NLRP3 inflammasome activation in fibrotic livers. ( A ) CD11b immunofluorescence staining with quantification of positive cells per high-power field. ( B ) Ly6G IHC and corresponding quantification. ( C–D ) Quantification of positive cells per high-power field (HPF). ( E ) mRNA levels of IL-1β, TNF-α, IL-6, and IL-10 were analyzed through qRT-PCR within liver tissues of mouse liver fiber models. ( F ) Measurement of serum IL-1β, TNF-α, and IL-6 levels within mice measured by ELISA. ( G–I ) protein expression of ASC, NLRP3, Pro-IL-1β, and IL-1β were carried out via Western blotting in liver tissues of mouse liver fiber models concerning the loading control GAPDH. ( J–M ) Liver sections from mice were exposed to NLRP3 and ASC IHC analysis, and the proportions of NLRP3- and ASC-positive regions were quantified. The data are expressed as the means ± SEMs; n = 6 per group; original magnification, 20×; scale bars, 100 μm; ∗ P < .05, ∗∗ P < .01.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Activation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: Nogo-B deficiency inhibits NLRP3 inflammasome activation in macrophages in vitro. ( A ) Nogo-B mRNA expression in BMDMS extracted from Nogo-B fl/fl and Nogo-B mko mice was identified using qRT-PCR. ( B ) Nogo-B protein expression within BMDMs extracted from Nogo-B fl/fl and Nogo-B mko mice was performed by Western blotting, GAPDH served as the loading control. ( C ) Western blotting analysis of Nogo-B protein in LPS-stimulated BMDMs from Nogo-B fl/fl and Nogo-B mKO mice, with GAPDH as the loading control. ( D ) mRNA levels of IL-1β, TNF-α, IL-6, and IL-10 were quantified in LPS-stimulated Nogo-B fl/fl and Nogo-B mko BMDMs by qRT-PCR. ( E ) Western blotting was used for exploring the levels of ASC, NLRP3, Pro-IL-1β, and IL-1β in LPS-stimulated Nogo-B fl/fl and Nogo-B mko BMDMs, GAPDH was the loading control. ( F ) IF staining for NLRP3 ( red ) in LPS-stimulated and unstimulated Nogo-B fl/fl and Nogo-B mko BMDMs. ( G ) IF staining for ASC ( red ) in LPS-stimulated and unstimulated Nogo-B fl/fl and Nogo-B mko BMDMs. ( H ) Western blotting was adopted for exploring the levels of ASC, NLRP3, Pro-IL-1β, and IL-1β in LPS-stimulated Nogo-B fl/fl and Nogo-B mko KCs, GAPDH served as the loading control. ( I ) Schematic drawing showed LX-2 cells exposed to treatment with CM of LPS-treated Nogo-B fl/fl and Nogo-B mko BMDMs. ( J ) IF staining for a-SMA ( red ) in LX-2 cells subject to treatment with CM of LPS-treated Nogo-B fl/fl and Nogo-B mko BMDMs. ( K ) Western blotting detected the expression of α-SMA, Collagen I, and TGF-β1 in LX-2 cells exposed to CM from LPS-treated Nogo-B fl/fl and Nogo-B mko BMDMs. ( L ) Schematic drawing showed that Nogo-B fl/fl and Nogo-B mko BMDMs were cocultured with LX-2 cells with or without LPS stimulation. ( M ) Transwell assay analyzed the migratory ability of LX-2 cells (original magnification 40×; scale bars, 50 μm). Data were shown to be mean ± SEM; ∗ P < .05, ∗∗ P < .01.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Control, Staining, Transwell Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: RIPK3 is critical for Nogo-B-facilitated NLRP3 inflammasome activation in macrophages in vivo and in vitro. ( A ) An IP‒MS workflow identified Nogo-B-interacting proteins. ( B ) Co-IP demonstrated an interaction between Nogo-B and RIPK3 in LPS-stimulated Nogo-B fl/fl BMDMs. ( C ) IF staining revealed Nogo-B ( red ) and RIPK3 ( green ) colocalization within LPS-stimulated Nogo-B fl/fl BMDMs. ( D ) IF staining revealed the colocalization of RIPK3 ( green ) and NLRP3 ( red ) and detected the expression of RIPK3 and NLRP3 in LPS-stimulated Nogo-B fl/fl and Nogo-B mko BMDMs. ( E ) Dual-IF staining of F4/80 ( green ) and RIPK3 ( red ) in liver tissues of mouse liver fiber models (n = 6 per group; original magnification 20×; scale bars, 100 μm). ( F ) Western blotting analysis measured RIPK3 in Nogo-B-deficient macrophages treated with Lv-RIPK3 or the RIPK3 inhibitor RIPK3-IN-4. ( G ) IF was used to determine the level of RIPK3 in LPS-stimulated Nogo-B mko BMDMs transfected with Lv-RIPK3 or RIPK3-IN-4. ( H ) qRT‒PCR was used to quantify IL-1β, TNF-α, IL-6, and IL-10 mRNA in LPS-stimulated Nogo-B mko BMDMs after Lv-RIPK3 transduction or RIPK3-IN-4 treatment. ( I ) IF staining for NLRP3 ( red ) in LPS-stimulated Nogo-B mko BMDMs transfected with Lv-RIPK3 or treated with RIPK3-IN-4. ( J ) IF staining for ASC ( red ) in LPS-stimulated Nogo-B mko BMDMs transfected with Lv-RIPK3 or RIPK3-IN-4 treatment. ( K ) Western blotting was used to detect ASC, NLRP3, pro-IL-1β, and mature IL-1β in LPS-stimulated Nogo-B mko BMDMs following Lv-RIPK3 or RIPK3-IN-4 treatment. The data are shown as the means ± SEMs; original magnification, 40×; scale bar = 50 μm; ∗∗ P < .01.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Activation Assay, In Vivo, In Vitro, Co-Immunoprecipitation Assay, Staining, Expressing, Western Blot, Transfection, Transduction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: RIPK3 is core to Nogo-B-induced necroptosis of macrophages both in vivo and in vitro. ( A ) Double fluorescence of necroptosis in LPS-stimulated Nogo-B fl/fl and Nogo-B mko BMDMs (original magnification, 40×; scale bars, 50 μm). ( B ) Flow cytometry was used to evaluate the viability of LPS-stimulated and unstimulated Nogo-B fl/fl and Nogo-B mko BMDMs. ( C ) Western blotting was performed to evaluate p-RIPK3, RIPK3, p-MLKL, MLKL, C-caspase-1, and Caspase-1 expression in LPS-stimulated and unstimulated Nogo-B fl/fl and Nogo-B mko BMDMs. ( D ) Liver sections from the mice were subjected to p-RIPK3 and p-MLKL IHC analysis. ( E ) The proportions of p-RIPK3-positive and p-MLKL-positive regions were quantified. ( F ) Western blotting was used to evaluate p-RIPK3, RIPK3, p-MLKL, MLKL, C-caspase-1, and Caspase-1 expression in the liver tissues of the mouse liver fiber models. ( G ) Western blotting was used to evaluate p-RIPK3, RIPK3, p-MLKL, MLKL, C-caspase-1, and Caspase-1 expression. ( H ) Flow cytometry was conducted to evaluate the cell viability of LPS-triggered Nogo-B mko BMDMs after Lv-RIPK3 or RIPK3-IN-4 transfection. The data are presented as the means ± SEMs; n = 6 per group; original magnification, 10×; scale bars, 200 μm; ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: In Vivo, In Vitro, Fluorescence, Flow Cytometry, Western Blot, Expressing, Transfection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: The adoptive transfer of BMDMs revealed that the Nogo-B/RIPK3 axis promoted NLRP3 inflammasome activation in macrophages and accelerated liver fibrosis. ( A ) Schematic illustration of adoptive transfer of BMDMs from mice. ( B ) ALT and AST levels in the serum of mice. ( C ) liver sections from mice subjected to Masson staining, Sirius Red staining, and α-SMA IHC analysis (original magnification 10×; scale bars, 200 μm). ( D ) Collagen I and α-SMA mRNA expression in murine livers was examined through qRT-PCR. ( E ) mRNA expression of IL-1β, TNF-α, IL-6, and IL-10 was quantified in liver tissues. ( F ) Protein expression of ASC, NLRP3, Pro-IL-1β, and IL-1β was measured via Western blotting in liver tissues. Data are presented as the means ± SEMs; n = 6 per group; ∗∗ P < .01. ∗ P < .05.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Adoptive Transfer Assay, Activation Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Macrophage Nogo-B Drives Liver Fibrosis

doi: 10.1016/j.jcmgh.2025.101622

Figure Lengend Snippet: Differences in Nogo-B + RIPK3 + macrophage populations within liver tissue after adoptive transfer of BMDMs. ( A ) Western blotting was used to evaluate the levels of p-RIPK3, RIPK3, p-MLKL, MLKL, C-caspase-1, and Caspase-1 in liver tissues. ( B–D ) Multiplex IF microscopy demonstrated triple colocalization of the macrophage markers F4/80 ( yellow ), Nogo-B ( green ), and RIPK3 ( red ) in fibrotic liver sections from mice receiving BMDMs in 3 independent models: ( B ) CCl 4 -induced, ( C ) BDL-induced, and ( D ) MCD-induced fibrosis. Nuclei were counterstained with DAPI ( blue ). n = 6 per group; original magnification, 20×; scale bars, 100 μm.

Article Snippet: The membranes were probed with the following primary antibodies: Nogo-B (rabbit mAb, Thermo Fisher), USP14, p-RIPK3, and p-MLKL (rabbit mAbs, Abcam); α-SMA, collagen I, TIMP1, MMP9, caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β, GAPDH, RIPK3, MLKL, CD11b, and LY6G (rabbit mAbs, Cell Signaling Technology); RIPK3 (mouse mAb, Cell Signaling Technology); USP14, NLRP3, and ASC (mouse mAbs, Proteintech); and Trim28 and Prpf19 (rabbit mAbs, Proteintech).

Techniques: Adoptive Transfer Assay, Western Blot, Multiplex Assay, Microscopy