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

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 1. Expression of mRNA coding for molecules of the Wnt signaling pathway in human Mu¨ller stem cells (hMSC) and modulation of WNT2B expression by transforming growth factor-b (TGFb1). (A) hMSC express mRNA coding for various components of the canonical and noncanonical Wnt signaling pathway. (B) TGFb1 downregulation of the expression of mRNA coding for WNT2B occurred in a dose–response manner in three different hMSC lines (MIO-M8, MIO-M7, and MIO-M1) after 7 days culture with concentrations of this cytokine ranging between 0.1 and 100ng/mL. Histograms represent the mean – standard error of the mean (SEM) from UV spectrophotometer readings of gel bands. Representative bands are shown below the histograms; n =3–4. ANOVA test, *P<0.05; **P< 0.01; ***P< 0.001. (C) A significant decrease in the expression of WNT2B protein was observed by western blot analysis of lysates from cells cultured with 50ng/mL of TGFb1. Histograms represent the mean– SEM of the relative optical density readings of gel bands. Representative bands are shown above the histograms; n= 3. Student’s t-test; *P<0.05. Minimally detectable levels of secreted WNT2B examined by Enzyme-Linked Immunosorbent Assay (ELISA) methods were observed in supernatants of cells cultured in the presence or absence of TGFb1, and no differences between the two conditions were observed; n =3. Student’s t-test; ns, not significant.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: Expressing, Spectrophotometry, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 3. Modulation of pb-catenin and DKK1 protein ex- pressions by TGFb1 and effect of exogenous WNT2B and WNT5B ligands on DKK1 mRNA expression. (A) Western blot analysis revealed that culture of hMSC with 50ng/mL of TGFb1 induced a significant upregulation of the ratio of phospho-b-catenin/b-catenin. Histograms represent the mean–SEM of the relative optical density readings of gel bands. Representative bands are shown above the histograms; n=5. Student’s t-test, *P <0.05. pb-catenin=phospho-b- catenin. (B) TGFb1 caused a significant decrease in DKK1 mRNA expression in hMSC as revealed by RT-PCR analysis. Histograms represent the mean–SEM from UV spectropho- tometer readings of gel bands. Representative bands are shown above the histograms; n =8. Student’s t-test; ***P< 0.001. Secreted DKK1 protein levels as determined by ELISA methods were significantly decreased in culture supernatants of cells treated with 50ng/mL of TGFb1 as compared to controls; n=4. Student’s t-test; *P<0.05. (C) Exogenous addition of recombinant WNT2B into the culture medium induced a sig- nificant upregulation of DKK1 mRNA in hMSC; n= 4. Stu- dent’s t-test, *P< 0.05. (D) Addition of recombinant WNT5B to cells in culture caused a significant downregulation of DKK1 mRNA expression; n =4. Student’s t-test, *P <0.05. RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Recombinant, Reverse Transcription, Polymerase Chain Reaction

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 4. Induction of photoreceptor differentiation by FTRI causes changes in the expression of the Wnt signaling components WNT2B, b-catenin, and DKK1 in hMSC. (A) Culture of hMSC with FTRI for 7 days caused a significant increase in the expression of WNT2B mRNA, while no changes in the expression of WNT5B were observed in these cells. Histograms represent the mean–SEM from UV spectrophotometer readings of gel bands. Representative bands are shown above the histograms; n=5. Student’s t-test, ***P <0.001. (B) Quantification of the secreted ligands, as measured by ELISA, showed that both WNT2B and WNT5B were significantly increased in culture supernatants of hMSC treated with FTRI for 7 days; n =3. Student’s t-test, *P <0.05. (C) Western blotting analysis showed that the ratio of phospho-b-catenin/b-catenin was decreased by FTRI treatment of hMSC. Histograms represent the mean– SEM of the relative optical density readings of gel bands. Representative bands are shown above the histograms; n=5. Student’s t-test, *P<0.05. pb-catenin=phospho-b-catenin. (D) A significant increase in the expression of DKK1 mRNA was observed in hMSC cultured with FTRI for 7 days; n=3. Student’s t-test, *P<0.05. ns, not significant; FTRI, FGF2, taurine, retinoic acid and insulin-like growth factor type1.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: Expressing, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 5. Inhibition of FTRI- induced photoreceptor differentia- tion of hMSC by TGFb1. (A) Culture of MIO-M1 cells with FTRI caused an increase in WNT2B mRNA ex- pression, but addition of TGFb1 to the differentiation medium inhibited this increase; n = 4. ANOVA test, **P < 0.01; ***P < 0.001. FTRI alone did not modify WNT5B mRNA expression, but addition of TGFb1 to the differentiation cocktail increased WNT5B mRNA expression (similar to that shown above with TGFb1 alone). Histograms represent the mean– SEM from UV spectropho- tometer readings of gel bands. Re- presentative bands are shown above the histograms; n = 3. ANOVA test, **P < 0.01; ***P < 0.001. (B) Addi- tion of TGFb1 to hMSC undergoing photoreceptor differentiation with FTRI inhibited the mRNA expres- sion of NR2E3, recoverin, and rho- dopsin as compared with FTRI alone; n = 5–8. ANOVA test, *P < 0.05; ***P < 0.001. (C) Im- munostaining for NR2E3 and re- coverin confirmed that while FTRI alone causeda markedincreaseinthe expression of this photoreceptor protein, addition of TGFb1 to hMSC cultured with FTRI caused inhibition of photoreceptor differentiation (Alexa 488, fluorescent cells). Cell nuclei counterstained with DAPI (non-fluorescent cell structures). Scale bars 50 mm. Histograms on the right represent the proportion of cells immunostaining for each of the markers following 7-day culture under the different conditions; n = 3. ANOVA test, **P < 0.01.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: Inhibition, Expressing, Cell Culture, Immunostaining

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 6. Effect of TGFb1 inhibitors on the expression of the Wnt signaling ligands WNT2B and WNT5B by hMSC. (A) Addition of the TGFb type I receptor (ALK5) inhibitor SB431542 (10 mM) to cells cultured with TGFb1 antago- nized the inhibitory effects of this cytokine on WNT2B mRNA expression; n = 5. ANOVA test; *P < 0.05; **P < 0.01. In contrast, addition of the JNK inhibitor SP600125 (20 mM) to cells cultured in the presence of TGFb1 did not modify the effect of this cytokine on WNT2B gene expression. Histograms represent the mean– SEM from UV spectrophotometer read- ings of gel bands. Representative bands are shown above the histograms; n = 3. ANOVA test, **P < 0.01. (B) While the ALK5 inhibitor SB431542 antagonized the upregulation of WNT5B mRNA by TGFb1; n = 3. ANOVA test; *P < 0.05; **P < 0.01, the JNK inhibitor SP600125 did not modify the effects of this cytokine on the expression of this ligand gene; n = 4. ANOVA test, **P < 0.01; ns, not significant.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Gene Expression, Spectrophotometry

Journal: Stem cells and development

Article Title: Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics.

doi: 10.1089/scd.2015.0262

Figure Lengend Snippet: FIG. 8. TGFb1 inhibits the canonical Wnt signaling path- way necessary for the photoreceptor differentiation of hMSC in vitro. Schematic illustration summarizing the interactions of FTRI, WNT2B, WNT5B, TGFb1, and DKK1 in hMSC. FTRI, which induces photoreceptor differentiation of hMSC, activated the canonical Wnt signaling pathway in these cells. Addition of TGFb1 to cells cultured with FTRI caused inhi- bition of the canonical Wnt signaling and consequently in- hibited the photoreceptor differentiation of hMSC in vitro.

Article Snippet: Supernatants were collected and used for Enzyme-Linked Immunosorbent Assay (ELISA) analysis for quantification of secreted DKK1 (R&D Systems), WNT2B, and WNT5B (CUSABIO) using the manufacturer’s instructions.

Techniques: In Vitro, Cell Culture

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