Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Immunohistochemistry staining of IL11RA and IL6R in healthy human liver sections (scale bars, 20 µm, n = 1 independent experiment, due to limited amount of human liver section). b Flow cytometry forward scatter (FSC) plots of IL11RA, IL6R, and gp130 staining and fluorescence intensity plots of IL11RA and IL6R staining on hepatocytes and THP-1. c Abundance of IL11RA1 and IL6R reads in hepatocytes at baseline based on RNA-seq (left) and Ribo-seq (right) (transcripts per million, TPM) ( n = 3). d , e Read coverage of d IL11RA1 and e IL6R transcripts based on RNA-seq (gray) and Ribo-seq (red) of primary human hepatocytes ( n = 3). f Western blots showing ERK, JNK, and STAT3 activation status and g ALT secretion ( n = 4) by hepatocytes following a dose range stimulation of either hyperIL11 or hyperIL6. h ALT levels in the supernatants of hepatocytes stimulated with hyperIL11 alone or in the presence of increasing amounts of soluble gp130 (sgp130) ( n = 4). i , j Western blots of hepatocyte lysates showing i phosphorylated ERK and JNK and their respective total expression in response to hyperIL11 stimulation alone or with sgp130 and j phospho-STAT3 and total STAT3 in response to hyperIL6 stimulation with and without sgp130. k Representative FSC plots of propidium Iodide (PI) staining of IL11-stimulated hepatocytes in the presence of sgp130 or soluble IL11RA (sIL11RA). l Western blots showing phospho-ERK, phospho-JNK, cleaved caspase-3, and their respective total expression, NOX4, and GAPDH in hepatocytes in response to IL11 stimulation alone or in the presence of sgp130 or sIL11RA. i , j , l Representative data of n = 2 independent experiments. b – l Primary human hepatocytes; f – l 24 h stimulation; hyperIL11, hyperIL6, IL11 (20 ng/ml), sgp130, sIL11RA (1 µg/ml). c , g , h Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). g , h One-way ANOVA with Dunnett’s correction. Source data are provided as a Source data file.

Article Snippet: Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin.

Techniques: Immunohistochemistry, Staining, Flow Cytometry, Fluorescence, RNA Sequencing, Western Blot, Activation Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a – l Data for palmitate (0.5 mM) loading experiment on primary human hepatocytes (24 h) in the presence of either IgG (2 µg/ml), anti-IL11RA (X209, 2 µg/ml), or sgp130 (1 µg/ml). a IL11, b IL6, c CCL2, and d CCL5 protein secretion levels as measured by ELISA of supernatants ( n = 3). e Representative FSC plots and f quantification of PI +ve hepatocytes stimulated with palmitate ( n = 3). g ALT levels in supernatants ( n = 3). h Total and reduced hepatocyte glutathione (GSH) levels ( n = 4). i Representative fluorescence images of DCFDA (2′,7′-dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100 µm) ( n = 4 independent experiments). j Western blots of phospho-ERK, ERK, phospho-JNK, JNK, cleaved caspase-3, caspase-3, NOX4, and GAPDH. Data from two independent biological experiments are shown. k Percentage of fatty acid oxidation by Seahorse assay ( n = 10). l Representative fluorescence images (scale bars, 100 µm) of ACTA2 +ve cells and Collagen I immunostaining for experiment shown in Supplementary Fig. ( n = 2 independent experiments, 14 measurements per condition per experiment). a – d , f , g Mean ± SD; h , k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). a – d , f – h , k One-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin.

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Staining, Western Blot, Immunostaining, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Schematic of WDF feeding in mice with hepatocyte-specific expression of sgp130 for data shown in ( b – p ). Three weeks following AAV8-Alb-Null or AAV8-Alb-sgp130 virus injection, mice were fed WDF for 16 weeks. b Western blots showing hepatic levels of sgp130, IL11, IL6, and GAPDH as internal control ( n = 4 mice/group). c Serum IL11 levels. d Serum IL6 levels. e Representative gross anatomy, H&E-stained (scale bars, 50 µm), and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 8 mice/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. f Liver weight. g Hepatic triglycerides content. h Serum ALT levels. i Serum AST levels. j Hepatic collagen levels. k Fasting blood glucose levels. l Serum triglycerides levels. m Serum cholesterol levels. n Hepatic GSH content. o Hepatic pro-inflammatory and fibrotic genes expression heatmap (values are shown in Supplementary Fig. and e). p Western blots of hepatic phospho-ERK, ERK, phospho-JNK, JNK, phospho-STAT3, and STAT3 ( n = 4 mice/group). c , d , f – o n = 8 mice/group. c , d , f – n Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); one-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin.

Techniques: Expressing, Virus, Injection, Western Blot, Control, Staining, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: a Schematic of HFMCD feeding regimen for AAV8-Alb-Cre injected Il11ra1 loxP/loxP (conditional knockout; CKO) mice for experiments shown in ( b – k ). Il11ra1 loxP/loxP mice were intravenously injected with either AAV8-Alb-Null or AAV8-Alb-Cre to delete Il11ra1 specifically in hepatocytes 3 weeks prior to the start of HFMCD diet. b Western blots of hepatic IL11RA and GAPDH ( n = 3 mice/group). c Body weight (shown as a percentage (%) of initial body weight). d Representative gross anatomy, H&E-stained (scale bars, 50 µm), and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 5 mice/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. e Hepatic triglycerides content. f Serum ALT levels. g Serum AST levels. h Hepatic GSH content. i Hepatic collagen levels. j Heatmap showing hepatic mRNA expression of pro-inflammatory markers ( Tnfα , Ccl2 , Ccl5 ) and fibrotic markers ( Col1a1, Col1a2, Col3a1, Acta2) . Values are shown in Supplementary Fig. and d. k Western blots showing hepatic ERK and JNK activation status ( n = 3 mice/group). c , e – j NCD ( n = 5 mice/group), HFMCD ( n = 6 mice/group). c Data are shown as mean ± SD, two-way ANOVA with Tukey’s correction, statistical significance ( P values) are shown for comparison between WT HFMCD and CKO HFMCD; e – i data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); two-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin.

Techniques: Injection, Knock-Out, Western Blot, Staining, Expressing, Activation Assay, Comparison, Whisker Assay

Journal: Nature Communications

Article Title: Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH

doi: 10.1038/s41467-020-20303-z

Figure Lengend Snippet: Excessive lipid accumulation in hepatocytes stimulates IL11 protein secretion and autocrine IL11 activity, which upregulates NOX4 and increases reactive oxygen species production. Subsequently, hepatocyte mitochondrial oxidative capacity and fatty acid metabolism impaired and steatosis established. ERK, JNK, and caspase-3 become activated and this leads to lipoapoptosis, along with other forms of cell death. IL11 also acts in paracrine to drive hepatic stellate cell-to-myofibroblast transformation and fibrosis. Cytokines and chemokines released from lipotoxic hepatocytes and HSCs activate and recruit immune cells causing inflammation.

Article Snippet: Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin.

Techniques: Activity Assay, Transformation Assay

Journal: ACS nano

Article Title: A Biomimetic Multifunctional Nanoframework for Symptom Relief and Restorative Treatment of Acute Liver Failure.

doi: 10.1021/acsnano.4c00173

Figure Lengend Snippet: Figure 5. Effects of RMBN on hepatic inflammation in LPS/GalN-induced murine models of ALF. (A) Levels of TNF-α, IL-6, IL-1β, MCP-1, and IL-17A involved in liver inflammation were analyzed by ELISA in the liver (n = 7 mice). ###P < 0.005 (light blue), Model vs CON groups. *P < 0.05, **P < 0.01, ***P < 0.005, and nsP > 0.05 (orange), compared with the RMBN group. ∧∧∧P < 0.005 (purple blue), RMBN vs Model groups. (B, C) Immunoblot analysis of factors in NLRP3-reated signaling pathways include NLRP3, IL-18, ASC, pro-IL-1β, and IL- 1β in the liver. (D) Heatmap of the differential expressed genes related to the NLRP3 inflammasome activation in the livers of mimic ALF mice with or without the RMBN (n = 4). RMBN + LPS/GalN vs LPS/GalN groups. Colors represent log2-transformed count (log2) change after normalization. (E, F) Immunoblot analysis of factors in NLRP3-reated signaling pathways include NLRP3, IL-18, ASC, pro-IL-1β, and IL-1β in the liver. (G) Representative immunofluorescence staining of CD206+ (red) and iNOS (green) in mouse liver sections. Scale bar = 50 μm. Data are expressed as means ± SD (n = 3−7). *P < 0.05, **P < 0.01, ***P < 0.005, and nsP > 0.05 compared with the vehicle group.

Article Snippet: Mouse CXCL12 ELISA Kit, Mouse IGF-1 ELISA Kit, and Mouse HGF ELISA Kit were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Protein-Protein interactions, Activation Assay, Transformation Assay, Immunofluorescence, Staining

Journal: Stem Cell Research & Therapy

Article Title: Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

doi: 10.1186/s13287-018-1056-1

Figure Lengend Snippet: HGFA is sufficient to induce the G ALERT transition and the functional molecular changes of BST2 pos CH cells. a Serum samples were prepared from non-injured (naive) or 24 h injured (fractured) mice, and HGFA levels were measured by ELISA. Bar graphs represent mean ± SD ( t value = 4.62, df = 6). ** p = 0.0036. The serum from four mice/group have been analyzed. b Schematic depicting the timelines of BM-derived BST2 pos CH cell isolation from fractured or recombinant HGFA (rHGFA)-injected mice contemporary receiving consecutive BrdU injections. ( c ) BM-derived BST2 pos CH cells derived from fractured (gray bar) or rHGFA-injected (red bar) mice have higher propensity to cycle in vivo than cells extracted from naive control mice (black bar). Three mice/experimental group have been used and four independent replicates have been performed. Bar graphs represent mean ± SD ( t = 3.11, df = 4) * p = 0.0357. d–k The expression of the selected motility-associated genes Hgf ( d ), Fgf2 ( e ), Igf1 ( f ), Met ( g ), Dpp4 ( h ), Ezr ( i ), uPar ( j ), and Mmp9 ( k ) was confirmed by quantitative PCR analysis, comparing the gene expression profiles of BST2 pos CH cells isolated from PB- and BM-Naive (14 mice), PB- and BM-Fracture (12 mice), and PB- and BM-rHGFA mice (six mice). Bar graphs represent mean ± SD, * p < 0.0306, ** p < 0.0096, **** p < 0.0001

Article Snippet: Serum HGFA levels were measured using the Mouse Hepatocyte growth factor activator (HGFAC) ELISA kit (Cusabio Biotech).

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Isolation, Recombinant, Injection, In Vivo, Control, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Isolation

Journal: The Tohoku journal of experimental medicine

Article Title: Osteopontin as a key mediator for vasculogenic mimicry in hepatocellular carcinoma.

doi: 10.1620/tjem.224.29

Figure Lengend Snippet: Fig. 7. Immunohistochemical staining for VM in HCC and normal liver tissues. (A) VM expression was identified in a HCC lesion by laminin immunostain (red arrow). (B) The same HCC lesion in (A) showing VM expression by laminin im- munostain did not express the endothelial cell marker CD31. Hepatic sinusoids were CD34 positive (yellow arrow). (C) The same HCC lesion in (A) was positive for hepatocyte. (D) A HCC lesion without VM expression by laminin im- munostain. Bars = 50 μm.

Article Snippet: After washing in 0.1 M phosphate-buffered saline (PBS), pH 7.4, and exposure to 3% normal goat serum for 20 min to reduce non-specific binding, the slides were incubated for 2 h at 37°C with a 1:100 dilution of anti-laminin monoclonal antibody (Millipore), or a 1:100 dilution of anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a working solution of anti-hepatocyte antibody (Zhongshan Goldenbridge Biotechnology CO Ltd, ready to use), or a 1:300 dilution of anti-OPN antibody (Santa Cruz), or a 1:300 dilution of anti-MMP-2 antibody (Santa Cruz), or a 1:300 dilution of anti-MMP-9 antibody (Santa Cruz), or a 1:300 dilution of anti-uPA antibody (Santa Cruz).

Techniques: Immunohistochemical staining, Staining, Expressing, Marker

Journal: Cell Death & Disease

Article Title: MANF serves as a novel hepatocyte factor to promote liver regeneration after 2/3 partial hepatectomy via doubly targeting Wnt/β-catenin signaling

doi: 10.1038/s41419-024-07069-8

Figure Lengend Snippet: Gene-specific primer sequences used for qPCR.

Article Snippet: The cells were plated on collagen-coated 6-well plate at a density of 2×10 5 cells/well, and treated with HGF (MCE, HY-P73928, 100 ng/ml), and then recombinant human MANF (10 μg/mL) was added for 12 h.

Techniques: