Journal: Communications Biology

Article Title: Exogenous L-arginine increases intestinal stem cell function through CD90+ stromal cells producing mTORC1-induced Wnt2b

doi: 10.1038/s42003-020-01347-9

Figure Lengend Snippet: CD90 + stromal cells was cultured in medium supplemented with L-arginine (1 mM) or not for 72 h respectively. a qPCR of relative mRNA expression of Wnt2b , Wnt1 , Wnt3 , Wnt3a , Wnt5a , Wnt6 , R-spondin 1 , and Gremlin1 genes in CD90 + stromal cells. Expression show is relative to GAPDH gene, n = 6. b Immunostaining of Wnt2b (red), Vimentin (green), and DAPI (blue) in CD90 + stromal cells. Scale bar, 10 μm. Quantitation for Wnt2b was measured by MFI, n = 6. c The concentration of Wnt2b in CD90 + stromal cells culture medium was detected by ELISA, n = 6. d Immunostaining of Wnt2b (red), EdU (green) and DAPI (blue) in jejunum. Scale bar, 50 μm. Quantitation for Wnt2b was measured by MFI, n = 6. (e) The concentration of Wnt2b in the SI crypts was detected by ELISA, n = 6. f Organoid formation per Lgr5 + ISCs treated with/without mouse recombinant WNT2B protein (300 ng/ml), n = 6. A representative image of organoids at day 3 is shown (red arrow marks organoids). Scale bar, 200 μm. g Organoid formation per Lgr5 + ISCs treated with mouse recombinant WNT2B protein (100 ng/ml), L-arginine (1 mM), Wnt2b antibody (1 μg/ml), and their combinations in co-culture model, n = 6. A representative image of organoids at day 3 is shown (red arrow marks organoids). Scale bar, 200 μm. Data are the mean ± SD; comparisons performed with t -tests (two groups) or analysis of variance (ANOVA) (multiple groups). * P < 0.05, ** P < 0.01, *** P < 0.001. Results are representative of two or three independent experiments.

Article Snippet: WNT2B levels were measured using an ELISA kit (CUSABIO) according to the manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Immunostaining, Quantitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Co-Culture Assay

Journal: Communications Biology

Article Title: Exogenous L-arginine increases intestinal stem cell function through CD90+ stromal cells producing mTORC1-induced Wnt2b

doi: 10.1038/s42003-020-01347-9

Figure Lengend Snippet: CD90 + stromal cells were cultured in medium supplemented with L-arginine (1 mM) for 3 days. a qPCR of relative mRNA expression of Slc7a1 and Slc7a4 genes in CD90 + stromal cells treated with L-arginine or not. Expression show is relative to GAPDH gene, n = 3. b Immunostaining of Slc7a1 (red) and DAPI (blue) in CD90 + stromal cells treated with L-arginine or not. Scale bar, 10 μm. Quantitation for Slc7a1 was measured by MFI, n = 6. c , d Protein levels of GAPDH, mTOR, P-mTOR, P70S6K, P-P70S6K, 4EBP1, and P-4EBP1 were measured by western blotting assay in CD90 + stromal cells treated with L-arginine or not, n = 3. e Immunostaining of Wnt2b (red), Vimentin (green), and DAPI (blue) in CD90 + stromal cells treated with L-arginine (1 mM), MHY1485 (5 μM), Rapamycin (5 μM) and their combinations for 3 days. Scale bar, 10 μm. f The concentration of Wnt2b in CD90 + stromal cells was detected by ELISA, n = 3. Data are the mean ± SD; comparisons performed with t -tests (two groups) or analysis of variance (ANOVA) (multiple groups). * P < 0.05, ** P < 0.01, *** P < 0.001. Results are representative of two or three independent experiments.

Article Snippet: WNT2B levels were measured using an ELISA kit (CUSABIO) according to the manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Immunostaining, Quantitation Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Development (Cambridge, England)

Article Title: RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling.

doi: 10.1242/dev.096990

Figure Lengend Snippet: Fig. 1. Signals involved in early optic vesicle patterning in the chick. (A) At stage HH 9, MITF protein is not detected in the ov (arrows). (B) At stage 10, MITF protein is present throughout the distal ov (arrow). Mesenchymal cells (arrowheads) are located dorsally. (C,C′) At stage 10, pSmad1/5 is detected in the entire distal ov (arrow). (D,D′) Higher magnification of C, showing pSmad1/5 labelling in neuroepithelial cells (arrow) and in the surface ectoderm (arrowhead). (E,E′) Stronger accumulation of pSmad1/5 is observed in the dorsal ov (arrows) compared with the ventral region (asterisks). (F,F′) Parallel section of E. Strong β- catenin labelling in the entire surface ectoderm/prospective lens ectoderm (arrow). Nuclear β-catenin is not observed in the ov (arrowhead). (G-I) Wnt2b expression during the initial stages of eye development. Wnt2b expression is restricted to the dorsal surface ectoderm (arrow), dorsal ov (arrowhead) and dorsal prosencephalon (white asterisks). Wnt2b transcripts are not detected in the ventral surface ectoderm and ov (asterisks). (J,K) Frontal and lateral view of a stage 10/11 chick head. Wnt2b transcripts in the ectoderm (arrows) covering the dorsal ov (white dashed line). Expression is not detected ventrally (asterisks). In all panels, dorsal is up.

Article Snippet: The DNA/RNA constructs used were Wnt2b siRNA and siRNA-A (both Santa Cruz), pC S2 hSmad1WT (Addgene plasmid 22989) and pC S2 hSmad1-GM (Addgene plasmid 22992; Edward de Robertis; Pera et al., 2003; Fuentealba et al., 2007).

Techniques: 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 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

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