Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b is elevated in hepatic fibrosis. ( a ) Representative Sirius Red staining, IHC staining of Wnt2b in liver tissue microarrays containing healthy controls (n = 9, with a mean age of 43.3 ± 1.4 years) and patients with fibrosis (n = 10, with a mean age of 48.8 ± 2.9 years). Hepatic fibrosis mouse model was induced by TAA, and then the following analyses were performed. ( b ) H&E, Sirius Red staining. ( c ) Immunofluorescence staining of α-SMA and IHC staining of Wnt2b. ( d ) RT-PCR (upper) and Western blotting (lower) of Wnt2b. Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . ( e ) ELISA analysis of Wnt2b in liver homogenate. Statistical analyses provided the mean ± SE (n = 8/group); * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b is mainly produced by hepatocytes during liver fibrogenesis. ( a ) mRNA and protein levels of Wnt2b in hepatocytes (left) and NPCs (right) from control and TAA-treated mice, respectively. ( b ) mRNA (upper) and protein (lower) levels of Wnt2b in primary mouse hepatocytes treated with CCl 4 in vitro . ( c ) Immunofluorescence staining of Wnt2b in HSCs from control and TAA-treated mice. ( d ) RT-PCR analysis of Wnt2b in cultured HSCs from naive mice at the indicated points of time. Mouse embryonic tissues served as positive controls. Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . Data are mean ± SEM of three independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Produced, Control, In Vitro, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b protects against liver fibrosis. ( a ) The schedule for Wnt2b knockdown with shRNA targeting mouse Wnt2b (sh-Wnt2b) or Wnt2b-overexpression with plasmid pRK5-mWnt2b (Over-Wnt2b) under TAA treatment. ( b ) The effects of sh-Wnt2b (left) and pRK5-mWnt2b (right) in TAA-challenged mice livers. ( c ) H&E and Sirius Red staining, Western blotting of Collagen-I, ( d ) Western blotting (upper) and immunofluorescence staining of α-SMA (lower) for liver tissues from mice treated with sh-Wnt2b or control vector. ( e ) H&E and Sirius Red staining, Western blotting of Collagen-I, ( f ) Western blotting (upper) and immunofluorescence staining of α-SMA (lower) for liver tissues from mice treated with pRK5-mWnt2b construct or control vector. Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . Statistical analysis are mean ± SE (n = 6/group); * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Knockdown, shRNA, Over Expression, Plasmid Preparation, Staining, Western Blot, Immunofluorescence, Control, Construct

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b exerts a direct inhibitory effect on HSCs activation. ( a ) RT-PCR analysis of Fzds and LRP5/6 in cultured HSCs from naive mice at the indicated points of time. Mouse embryonic tissues served as positive controls. ( b , c ) mRNA and protein levels of α-SMA and Collagen-I in LX2 cells cultured in conditioned medium (CM) collected from HEK293 cells transfected with active Wnt2B-V5 (CM-Wnt2b) or control plasmid (CM-Control) ( b ), as well as in LX2 cells transfected with active Wnt2B-V5 or control plasmid ( c ). Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . Data are mean ± SEM of three independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b suppresses TLR4 activation-mediated pro-fibrogenic effects. ( a ) Representative images for bacterial growth of jejunum (left) and liver tissues (right) after cultivation on Blood Agar Plates. ( b ) Representative H&E (upper) and Sirius Red staining (lower) of liver tissues from WT mice and TLR4 −/− mice after 12 intraperitoneal injections of TAA. ( c ) H&E and Sirius Red staining,Western blotting of α-SMA in liver tissues from mice treated with TAA alone, or combined with sh-Wnt2b construct/TLR4 inhibitor TAK242 (4 mg/kg, i.p.), or all of the three factors given above in combination for 4 weeks. ( d ) Protein levels of α-SMA and Collagen-I in LX2 cells stimulated with LPS (10, 100 ng/ml) for 24 h. ( e ) Effects of Wnt2b on the α-SMA and Collagen-I expressions in LX2 cells stimulated with LPS (100 ng/ml). ( f ) Protein levels of α-SMA and Collagen-I in LX2 cells stimulated with LPS (100 ng/ml) or vehicle for 24 h, and TGF-β (500 pg/ml) or vehicle for an additional 48 h. ( g ) LX2 cells were transfected with active Wnt2B-V5 or control plasmids for 24 h, followed by treatment with LPS ± TGF-β as described in Fig. 5f. The expression of α-SMA and Collagen-I were then detected by Western blot analysis. Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . Statistical analysis provided the mean ± SE (n = 6/group), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Activation Assay, Staining, Western Blot, Construct, Transfection, Control, Expressing

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: Wnt2b disturbs the TLR4 signaling transduction. ( a , b ) Protein levels of TLR4, p-NF-κB p65 (Ser536) and NF-κB p65 (upper), and mRNA levels of RELA and TNF (lower) in LX2 cells cultured in Wnt2b-CM ( a ), as well as in LX2 cells transfected with active Wnt2B-V5 or control plasmids ( b ). ( c , d ) Western blotting of the phosphorylation of MAPKs in LX2 cells cultured in Wnt2b-CM ( c) , and in LX2 cells transfected with active Wnt2B-V5 or control plasmids ( d ). ( e,f ) Protein levels of TLR4, p-NF-κB p65 (Ser536) and NF-κB p65 ( e ), and mRNA levels of RELA and TNF ( f ) in liver tissues from mice challenged with TAA combined with HD injection of pRK5-mWnt2b/sh-Wnt2b construct or control vector as depicted in Fig. . ( g ) Immunofluorescence staining of NF-κB p65 in HSCs isolated from mice challenged with TAA combined with HD injection of pRK5-mWnt2b (upper) / sh-Wnt2b (lower) construct or control vector as depicted in Fig. . Cropped blots are displayed; Full-length blots are presented in Supplementary Fig. . Statistical analysis provided the mean ± SE (n = 6/group), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Transduction, Cell Culture, Transfection, Control, Western Blot, Phospho-proteomics, Injection, Construct, Plasmid Preparation, Immunofluorescence, Staining, Isolation

Journal: Scientific Reports

Article Title: Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

doi: 10.1038/s41598-017-04374-5

Figure Lengend Snippet: A schematic model for the inhibitory effects of Wnt2b on HSCs activation and liver fibrosis through negative regulating TLR4 signaling.

Article Snippet: Active Wnt2B-V5 encoding homonine Wnt2b was a gift from Xi He (Addgene plasmid # 43808) .

Techniques: Activation Assay