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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

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: The shRNA plasmid targeting mouse Wnt2b was purchased from OriGene (Gene ID 22414, Beijing, China).

Techniques: Activation Assay

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 regulates injury-related expression of neuropeptides and other genes. ( a–i ) Unlesioned and injured FN of Atf3 +/+ and Atf3 −/− mice were subjected to qPCR analysis after 3 days of injury to quantify mRNA abundance of primers indicated. In wt mice (grey bars), facial nerve injury resulted in induction of Sprr2j ( a ), Vip ( b ), Ngf ( c ), Wnt2b ( d ), Galanin ( e ), Grp ( f ), Timp1 ( g ) and the known ATF3 target gene Hsp27 ( h ). In contrast to wt mice, induction of Sprr2j, Vip, Ngf, Wnt2b, Galanin, Grp and Hsp27 mRNA abundance was reduced upon facial nerve lesion in Atf3 mutant mice (white bars). Timp1 mRNA induction was more pronounced upon ATF3 loss-of-function ( g ). The Vip2 receptor ( Vipr2 ) was downregulated by facial nerve injury in wt and ATF3-deficient mice ( i ). Numbers in bars in ( b ) reflect independent biological replicates for experiments in ( a–i ). ( j–l ) Confirmation of reduced galanin expression in ATF3-deficient mice upon facial nerve injury. Deafferented wt ( j ) and ATF3-deficient ( k ) FN were stained with anti-galanin directed antibodies. In wt mice ( j ), galanin localized in secretory vesicle-like structures (see inset) of FMNs (some labelled with an arrow). The number of galanin immunoreactive FMNs was reduced in Atf3 mutant mice ( k ). ( l ) Quantification of galanin positive neurons without and 3 and 12 days after lesion. Data are presented as mean ± s.d. ** p ≤ 0.01. Scale bar ( j,k ) = 50 µm; inset = 5 µm.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Mutagenesis, Staining

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 mediates neuropeptide and Wnt2b expression in primary PNS neurons. ( a–d ) Adult wt or ATF3-deficient mouse DRG neurons were infected with adenoviral (AV) particles resulting in GFP (control) or ATF3 expression. mRNA levels of Atf3 ( a ), Wnt2b ( b ), Galanin ( c ) and Grp ( d ) were analysed by qPCR. Viral infection strongly enhanced Atf3 mRNA abundance in wt and Atf3 mutant neurons ( a ). Wnt2b ( b ), Galanin ( c ) and Grp ( d ) mRNA levels were augmented upon viral ATF3 overexpression in wt and Atf3 mutant DRG neurons. ( e,f ) Primary wt neurons overexpressing GFP or ATF3 were subjected to ChIP analysis with anti-ATF3 or IgG (control) antibodies. ATF3 occupancy at potential ATF3 binding sites of the Galanin ( e ) and Grp ( f ) promoter was tested with qPCR. ATF3 promoter occupancy was observed in ATF3-overexpressing samples only in the presence of anti-ATF3 but not IgG antibodies suggesting ATF3 binding at the Galanin ( e ) and Grp ( f ) promoter. Numbers in bars reflect independent numbers of experiments. Data are presented as mean ± s.d. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Infection, Control, Mutagenesis, Over Expression, Binding Assay