Journal: The Journal of Clinical Investigation

Article Title: Aortic carboxypeptidase–like protein, a WNT ligand, exacerbates nonalcoholic steatohepatitis

doi: 10.1172/JCI92863

Figure Lengend Snippet: (A, D–F) Eight-week-old male Aclpfl/fl and AclpHSC-KO mice were fed an HFC diet for 4 weeks (n = 5/group) or 24 weeks (n = 7/group) to prepare a murine model of NAFLD or were fed a control diet for 24 weeks (n = 6/group). (A) Serum levels of FFA, leptin, and IL-6. **P < 0.01 vs. Aclpfl/fl mice fed a control diet. (B) Quantification of hepatic Aclp mRNA in 8-week-old WT mice 4 hours after treatment with intralipid (n = 7/group), leptin (n = 7/group), IL-6 (n = 7/group), or PBS (n = 5/group). *P < 0.05; **P < 0.01 vs. control mice treated with PBS. (C) Quantification of Aclp mRNA in primary cultured murine HSCs treated with various combinations of palmitate (200 μM), leptin (100 ng/ml), and IL-6 (100 ng/ml) for 6 hours (n = 5–7/group). **P < 0.01 vs. control HSCs treated with PBS. (D–F) Liver sections from control and NAFLD model Aclpfl/fl mice were used. (D) (Upper panel) Representative images of immunofluorescence double-staining for p-STAT3 (red) and GFAP (green). Scale bars: 100 μm. Single-channel images are shown in Supplemental Figure 20. (Lower panel) Quantification of pSTAT3/GFAP double-positive cells. The nuclei were stained with DAPI (blue). p-STAT3/nucleus costained sites are shown in purple. **P < 0.01 vs. Aclpfl/fl mice fed a control diet. (E) (Upper panel) Representative immunofluorescence double-staining images of p-STAT3 (red) and ACLP (green). Scale bars: 100 μm. Single-channel images are shown in Supplemental Figure 21. (Lower panel) Quantification of pSTAT3/ACLP double-positive cells. The nuclei were stained with DAPI (blue). p-STAT3/nucleus costained sites are shown in purple. **P < 0.01 vs. Aclpfl/fl mice fed a control diet. (F) Correlation between p-STAT3/GFAP double-positive and ACLP-positive cells. P values obtained via 1-way ANOVA with Tukey’s post hoc test (A, C, D, and E), unpaired Student’s t tests (B), and Pearson’s correlation analysis (F). Data are shown as SEM.

Article Snippet: Recombinant mouse DKK-1, mouse leptin, mouse IL-6, and human FZD8/Fc were purchased from R&D Systems.

Techniques: Cell Culture, Immunofluorescence, Double Staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Aortic carboxypeptidase–like protein, a WNT ligand, exacerbates nonalcoholic steatohepatitis

doi: 10.1172/JCI92863

Figure Lengend Snippet: (A) Serum levels of FFA, leptin, and IL-6 in healthy control subjects (n = 20), NAFL patients (n = 16), and NASH patients (n = 44). (B–D) Liver sections from controls (n = 14), NAFL patients (n = 16), and NASH patients (n = 44) were used. (B) (Upper panel) Representative images of immunofluorescence double-staining of p-STAT3 (red) and GFAP (green). Scale bars: 100 μm. Single-channel images are shown in Supplemental Figure 23. (Lower panel) Quantification of pSTAT3/GFAP double-positive cells. The nuclei were stained with DAPI (blue). p-STAT3/nucleus costained sites are shown in purple. **P < 0.01 vs. control subjects. (C) (Upper panel) Representative immunofluorescence double-staining images of p-STAT3 (red) and ACLP (green). Scale bars: 100 μm. Single-channel images are shown in Supplemental Figure 23. (Lower panel) Quantification of pSTAT3/ACLP double-positive cells. The nuclei were stained with DAPI (blue). p-STAT3/nucleus costained sites are shown in purple. **P < 0.01 vs. control liver samples. (D) Correlation between p-STAT3/GFAP double-positive and ACLP-positive cells. P values obtained via 1-way ANOVA with Tukey’s post hoc test (A–C) and Pearson’s correlation analysis (D). Data are shown as SEM.

Article Snippet: Recombinant mouse DKK-1, mouse leptin, mouse IL-6, and human FZD8/Fc were purchased from R&D Systems.

Techniques: Immunofluorescence, Double Staining, Staining

Journal: Gut microbes

Article Title: Intestinal deguelin drives resistance to acetaminophen-induced hepatotoxicity in female mice.

doi: 10.1080/19490976.2024.2404138

Figure Lengend Snippet: Figure 5. The inhibition of TSHR confers effect of deguelin against APAP-induced hepatocyte oxidative stress. (a) Flow chart outlining the preparation of primary hepatocytes for RNA sequencing. The primary hepatocytes were pretreated with or without 0.1 μM deguelin, followed by treatment with 5 mM APAP for 3 h. (b) KEGG pathway enrichment analysis of the 74 DEGs with fold change greater than 4.

Article Snippet: The protocols were described as previously.24 Briefly, recombinant mouse TSHR protein (bs-42044P, Bioss, China) was immobilized on a Biacore 3D Dextran chip, and then activation with equal amounts of 0.49 M 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1 M N-hydroxy-succinimide.

Techniques: Inhibition, RNA Sequencing

Journal: Molecular Medicine Reports

Article Title: AKR1C1 alleviates LPS-induced ALI in mice by activating the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2021.12473

Figure Lengend Snippet: Correlation between AKR1C1 expression and serum levels of TNF-α, IL-6, IL-10 and MDA. In the lipopolysaccharide-induced acute lung injury mouse model, the correlation between the fold changes of AKR1C1 expression and serum levels of (A) TNF-α, (B) IL-6, (C) IL-10 and (D) MDA were analyzed using Pearson's correlation coefficient. n=12. AKR1C1, aldo-keto reductase family 1 member C1; IL, interleukin; TNF-α, tumor necrosis factor-α; MDA, malondialdehyde.

Article Snippet: Mice in the LPS+AKR1C1 +/+ +IL-6 group received intratracheal instillation of LPS and intravenous injection of a virus carrying the AKR1C1 vector and intratracheal instillation of recombinant mouse IL-6 (100 ng; BD Pharmingen; BD Biosciences) to activate the JAK2/STAT3 signaling pathway.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: AKR1C1 alleviates LPS-induced ALI in mice by activating the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2021.12473

Figure Lengend Snippet: Effect of AKR1C1 overexpression or knockout on LPS-induced inflammation. In the LPS-induced mouse model of acute lung injury, (A) total cell and neutrophil counts in BALF, and the levels of IL-6, IL-1 and TNF-α in (B) BALF and (C) lung tissues were measured in the wild-type mice (Control, LPS and LPS+E-virus), AKR1C1 overexpression mice (LPS+AKR1C1 +/+ ) andAKR1C1 knockout mice (LPS+AKR1C1 −/− ). # P<0.05 vs. Control; *P<0.05 vs. LPS+E-virus; & P<0.05 vs. LPS. n=12. LPS, lipopolysaccharide; AKR1C1, aldo-keto reductase family 1 member C1; IL, interleukin; TNF-α, tumor necrosis factor-α; BALF, bronchoalveolar lavage fluid; E-virus, empty lentiviral vector.

Article Snippet: Mice in the LPS+AKR1C1 +/+ +IL-6 group received intratracheal instillation of LPS and intravenous injection of a virus carrying the AKR1C1 vector and intratracheal instillation of recombinant mouse IL-6 (100 ng; BD Pharmingen; BD Biosciences) to activate the JAK2/STAT3 signaling pathway.

Techniques: Over Expression, Knock-Out, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: AKR1C1 alleviates LPS-induced ALI in mice by activating the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2021.12473

Figure Lengend Snippet: Effect of JAK2/STAT3 agonists on AKR1C1-mediated lung injury in the LPS-induced mouse model of acute lung injury. AKR1C1 +/+ mice were treated with LPS and JAK2/STAT3 agonists (IL-6 or colivelin). (A) Representative images of western blot analysis. Protein expression levels of (B) p-JAK2 and JAK2, and (C) p-STAT3 and STAT3 were semi-quantified. (D) Evans blue leakage in the lung, (E) lung wet/dry weight ratio, (F) PaO 2 /FIO 2 ratio, (G) survival rate of mice, and the oxidative products (H) protein carbonyl and (I) MDA in the lung were measured. # P<0.05 vs. Control; & P<0.05 vs. LPS; ^ P<0.05 vs. LPS+AKR1C1 +/+ . n=12. LPS, lipopolysaccharide; AKR1C1, aldo-keto reductase family 1 member C1; JAK, Janus kinase; STAT, signal transduction activator of transcription; p, phosphorylated; IL, interleukin; MDA, malondialdehyde.

Article Snippet: Mice in the LPS+AKR1C1 +/+ +IL-6 group received intratracheal instillation of LPS and intravenous injection of a virus carrying the AKR1C1 vector and intratracheal instillation of recombinant mouse IL-6 (100 ng; BD Pharmingen; BD Biosciences) to activate the JAK2/STAT3 signaling pathway.

Techniques: Western Blot, Expressing, Transduction

Journal: Theranostics

Article Title: Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling

doi: 10.7150/thno.94207

Figure Lengend Snippet: POSTN promotes the proliferation and IL-4 expression in IHH-4 cells by activating the integrin-FAK-STAT3 signaling. ( A ) Immunohistochemical analysis of the expression of IL-4 in tumor tissues from Postn +/+ Rag1 -/- and Postn -/- Rag1 -/- mice. Scale bars, 50 μm. ( B - D ) Western blot ( B ), qRT-PCR ( C ) (n = 4) and ELISA ( D ) (n = 6) analyses of IL-4 expression in tumor tissues of Postn +/+ Rag1 -/- and Postn -/- Rag1 -/- mice. ( E ) Immunohistochemical analysis of the expression of IL-4 and EpCAM in human papillary thyroid tumor. Scale bars, 50 μm. ( F and G ) Western blot ( F ) and qRT-PCR ( G ) analyses of IL-4 expression in IHH-4 cells cultured alone or co-cultured indirectly with WT or KO CAFs (n = 3). ( H and I ) Western blot ( H ) and ELISA ( I ) analyses of IL-4 level in IHH-4 cells treated with or without 100 ng/mL rhPOSTN (n = 6). ( J and K ) Western blot ( J ) and qRT-PCR ( K ) analyses of the indicated proteins and genes in IHH-4 cells treated with 100 ng/mL rhPOSTN in combination with antibodies against integrins αvβ3 or αvβ5 (n = 4). ( L ) Analysis of the proliferation of IHH-4 cells treated as in ( J ) for the indicated time by CCK8 assay (n = 4). ( M and N ) Western blot ( M ) and qRT-PCR ( N ) analyses of the indicated proteins and genes in IHH-4 cells treated with 100 ng/mL rhPOSTN alone or together with the indicated inhibitors (n = 3). ( O ) Analysis of the proliferation of IHH-4 cells treated as in ( M ) for the indicated time by CCK8 assay (n = 3). ( P )Western blot analysis of the indicated proteins in the tumor tissues of Postn +/+ Rag1 -/- and Postn -/- Rag1 -/- mice with papillary thyroid tumors. Data are shown as means ± SEM. Student's t test (C, D and I). One-way ANOVA (G, K and N). Two-way ANOVA (L and O). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., no significant difference.

Article Snippet: Similarly, CAFs were cultured with serum-free medium for 6 hours, before treatment with 25 ng/mL rmIL-4 (Abclonal, RPO1161) or recombinant human IL-4 protein (rhIL-4) (Abclonal, RP00995), CAFs were incubated with anti-IL-4R antibody (Abcam, ab271041, 5 μg/mL) or AS1517499 (500 nM) (MedChemExpress, HY-100614) for 30 minutes.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, CCK-8 Assay

Journal: Theranostics

Article Title: Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling

doi: 10.7150/thno.94207

Figure Lengend Snippet: IL-4 enhances the activation and POSTN expression in CAFs. ( A - C ) Western blot ( A ) and qRT-PCR ( B and C ) analyses of POSTN and Acta2 in CAFs cultured alone or co-cultured indirectly with IHH-4 or TPC-1 cells (n = 4). ( D and E ) Relative mRNA levels of Postn ( D ) and Acta2 ( E ) in isolated CAFs treated with rmIL-4 for the indicated time (n = 4). ( F and G ) Relative mRNA levels of Postn ( F ) and Acta2 ( G ) in isolated CAFs treated with rmIL-4 for the indicated doses for 24 hours (n = 3). ( H and I ) Western blot ( H ) and qRT-PCR ( I ) analyses of POSTN in CAFs treated with rmIL-4 (n = 3). ( J ) ELISA analysis of POSTN level in CAFs treated with rmIL-4 (n = 6). ( K and L ) Immunofluorescence images ( K ) and quantitation ( L ) of POSTN + cells in CAFs treated with or without rmIL-4. Scale bars, 50 μm (n = 6). ( M - O ) Western blot ( M and N ) and qRT-PCR ( O ) analyses of indicated proteins and Postn gene in CAFs treated with rmIL-4 alone or together with indicated inhibitors (n = 3). ( P ) Schematic representation of full length and two truncated luciferase reporters of mouse Postn promoter, and predicted binding sequences were predicted through a website-based search ( https://guolab.wchscu.cn/hTFtarget/ ). ( Q ) Relative luciferase assay showing the mouse Postn promoter activity after co-transfected with vector Stat6 or vector control in HEK 293T cells (n = 3). ( R ) Relative enrichment of Stat6 on the indicated regions of Postn promoter in IL-4-treated CAFs detected by ChIP-qPCR assay using anti-STAT6 (phospho Y641) or control IgG antibody (n = 3). Data are shown as means ± SEM. One-way ANOVA (B-G and O). Student's t test (I, J and L). Two-way ANOVA (Q and R). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., no significant difference.

Article Snippet: Similarly, CAFs were cultured with serum-free medium for 6 hours, before treatment with 25 ng/mL rmIL-4 (Abclonal, RPO1161) or recombinant human IL-4 protein (rhIL-4) (Abclonal, RP00995), CAFs were incubated with anti-IL-4R antibody (Abcam, ab271041, 5 μg/mL) or AS1517499 (500 nM) (MedChemExpress, HY-100614) for 30 minutes.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Quantitation Assay, Luciferase, Binding Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: Theranostics

Article Title: Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling

doi: 10.7150/thno.94207

Figure Lengend Snippet: The correlation between POSTN and IL-4 expression in human thyroid tumors. ( A and B ) The expression of POSTN and IL-4 in normal thyroid tissues (NT, n = 29), papillary thyroid cancer [PTC, n = 46] and anaplastic thyroid cancer [ATC, n = 10] was analyzed in the GSE33630 dataset. One-way ANOVA with multiple comparisons test. ( C ) Spearman correlation analysis of IL-4 with POSTN of human papillary thyroid cancer in the GSE33630 database (n = 46). Spearman correlation coefficient (R). ( D ) Spearman correlation analysis of POSTN expression with infiltration level of CAF of human thyroid cancer in the Timer 2.0 database. ( E ) Spearman correlation analysis of POSTN with STAT3 of human thyroid cancer in the GEPIA database. Normalized by HMBS. ( F ) Spearman correlation analysis of IL-4 with STAT6 of human thyroid cancer in the GEPIA database. Normalized by HMBS. ( G and H ) Western blot ( G ) and qRT-PCR ( H ) analyses of POSTN and IL-4 in clinical papillary thyroid tumors (n = 3). Two-way ANOVA with multiple comparisons test. ( I ) Representative immunohistochemical images of POSTN and IL-4 in clinical papillary thyroid tumors and their corresponding adjacent tissues. Scale bars, 50 μm. ( J ) Model indicating CAF-derived POSTN promotes papillary thyroid tumor cells to secrete IL-4 through the integrin-FAK-STAT3 signaling, which in turn IL-4 stimulates CAFs to produce POSTN via STAT6 activation. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Similarly, CAFs were cultured with serum-free medium for 6 hours, before treatment with 25 ng/mL rmIL-4 (Abclonal, RPO1161) or recombinant human IL-4 protein (rhIL-4) (Abclonal, RP00995), CAFs were incubated with anti-IL-4R antibody (Abcam, ab271041, 5 μg/mL) or AS1517499 (500 nM) (MedChemExpress, HY-100614) for 30 minutes.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Derivative Assay, Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: IL-6 and TNF-α have distinct effects on FLS and chondrocytes. ( a and b ) Mouse FLS ( a ) and chondrocytes ( b ) were infected with the indicated MOI of Ad- Epas1 , and the levels of secreted IL-6 and TNF-α in the culture media were determined by ELISA. ( c and d ) The indicated amounts of recombinant mouse IL-6 protein were used to treat FLS ( c ) or chondrocytes ( d ) for 24 h, and the mRNA levels of Mmp3 and Mmp13 were detected by qRT-PCR. ( e and f) Mouse FLS ( e ) and articular chondrocytes ( f ) were treated with the indicated doses of recombinant mouse TNF-α protein for 24 h, and the levels of the indicated catabolic factors were determined by qRT-PCR. Values are presented as means±s.e.m. (* P <0.01, ** P <0.005).

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: Blockade of IL-6 secretion in FLS or chondrocytes in co-culture. ( a ) Chondrocytes isolated from WT or Il6 −/− mice were infected with Ad- Epas1 and co-cultured with FLS for an additional 24 h. The expression levels of Epas1 , Il6 , Mmp3 , and Mmp13 were detected by RT-PCR and quantified by qRT-PCR. ( b ) FLS isolated from WT or Il6 −/− mice were infected with the indicated amounts (MOI) of Ad- Epas1 and then co-cultured with chondrocytes. RT-PCR and qRT-PCR were carried out to detect Epas1 , Il6 , Mmp3 , and Mmp13 in chondrocytes. Values are presented as means±s.e.m. (* P <0.05, ** P <0.005).

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Isolation, Infection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

doi: 10.1038/emm.2015.88

Figure Lengend Snippet: The receptors for IL-6 and TNF-α show distinct expression patterns in HIF-2α-overexpressing FLS and chondrocytes. ( a and b ) Mouse FLS or chondrocytes were infected with Ad- Epas1 or empty virus at an MOI of 800 for 24 h, and microarray analyses were performed on chondrocytes and FLS. Microarray data for IL-6 receptors ( Il6ra and Il6st ) and TNF-α receptors ( Tnfrsf1a and Tnfrsf1b ) in Ad- Epas1 -infected FLS ( a ) and chondrocytes ( b ) are shown. Transcript expression levels are shown relative to those in Ad-C-treated cells. ( c) Schematic diagram summarizing the regulatory mechanism governing the in vitro interaction between FLS and chondrocytes in the presence of HIF-2α overexpression.

Article Snippet: For isolation of chondrocytes, mouse cartilage tissues were isolated from the femoral condyles and tibial plateaus of wild-type and Il6 −/− mice, and chondrocytes were extracted by digestion with 0.2% collagenase type II, as described previously., Chondrocytes or FLS were treated with the indicated amounts of recombinant mouse IL-6 protein (Merck-Millipore, Billerica, MA, USA), recombinant mouse TNF-α protein (Merck-Millipore) or 800 MOI (multiplicity of infection) of empty adenovirus (Ad-C) or HIF-2α-expressing adenovirus (Ad- Epas1 ) for 24 h with or without subsequent treatment with the anti-TNF-α blocking antibody (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Expressing, Infection, Microarray, In Vitro, Over Expression