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: eLife

Article Title: Injury-induced perivascular niche supports alternative differentiation of adult rodent CNS progenitor cells

doi: 10.7554/eLife.30325

Figure Lengend Snippet: ( A ) Graphical representation of BMP/WNT/Hedgehog signalling pathway interactions based on 10 dpl microarray results, higher expression of particular gene in non-VN in red, a higher expression in a VN group in green. Computed expression changes were marked over the existing topology using green-red colour scheme. Box size represents the relative difference of expression, genes with significant changes of expression bolded (Fisher’s exact test, adj.p < 0.05). In order to highlight observed differential co-regulation of gene expression, consistent relations were marked with green. Two genes were assumed to be in consistent relation if changes of their expression levels between the compared groups were consistent with type of regulations between them (activation or inhibition). Such consistent relations indicate functionally related expression changes . ( B ) Absolute expression of BMP4 , Wnt2b , and Wnt6 in VN (green) and non-VN (red) at 6 and 10 dpl, dots represent expression level for each sample. ( C ) Relative gene expression (mean ± sd; n = 3, Mann-Whithney U-test, *p<0.001 compared with control levels of intact tissue as well as with non-VN at the same time point, # p<0.05 compared with control levels of intact, @p<0.01 compared with BMP4 level at 10 dpl, $p<0.001 compared with control levels of intact tissue as well as with 6 dpl and 10 dpl).

Article Snippet: We used the following primer/probe sets: Actb Rn00667869_m1; BMP4 Rn00432087_m1; CTGF Rn01537279_g1; Rn18s Rn03928990_g1; Sostdc1 Rn00596672_m1; Wnt2Rn01500736_m1; Wnt2b Rn00627297_m1.

Techniques: Microarray, Expressing, Gene Expression, Activation Assay, Inhibition, Control

Journal: eLife

Article Title: Injury-induced perivascular niche supports alternative differentiation of adult rodent CNS progenitor cells

doi: 10.7554/eLife.30325

Figure Lengend Snippet: ( A ) For in vitro expression analysis we used extra-pure populations of OPCs and astrocytes obtained by three times repeated shaking step since standard procedure of isolation results with astrocyte cultures highly enriched with OPCs. Images show cultures re-seeded after indicated shaking repetition (scale bar 100 μm). ( B ) qPCR results confirm that oligodendrocyte progenitors and endothelial cells produce BMP4 and Wnt2b while Sostdc1 is produced mainly by astrocytes in control cultures or at indicated time after scratch in wound healing assay (days post treatment, dpt). Data presented as means of ΔCT ± sd, one-way ANOVA, Newman-Keuls test, ***p<0.001.

Article Snippet: We used the following primer/probe sets: Actb Rn00667869_m1; BMP4 Rn00432087_m1; CTGF Rn01537279_g1; Rn18s Rn03928990_g1; Sostdc1 Rn00596672_m1; Wnt2Rn01500736_m1; Wnt2b Rn00627297_m1.

Techniques: In Vitro, Expressing, Isolation, Produced, Control, Wound Healing Assay

Journal: PloS one

Article Title: Differential effects of β-catenin and NF-κB interplay in the regulation of cell proliferation, inflammation and tumorigenesis in response to bacterial infection.

doi: 10.1371/journal.pone.0079432

Figure Lengend Snippet: Figure 4. Evidence of b-catenin and NF-kB interplay in vitro. A. Measurement of Wnt2b and Wnt5a expression in YAMC (Young Adult Mouse Colon) cells in vitro. YAMC cells (56105) were either uninfected (N) or infected with CR at 90:1 MOI for 3 hr. Cells were washed thoroughly to remove bacteria and incubated in fresh medium containing antibiotics for indicated period of time. Total RNA was examined for the expression of Wnt2b and Wnt5a via RT-PCR. GAPDH was used as loading control. B. Effect of Wnt2b knockdown on reporter activity. YAMC cells were transiently transfected with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 h, cells were infected with CR at 90:1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla luciferase as internal control (w, p,0.05 vs. N; ww, p,0.05 vs. CR; *, p,0.05 vs. CR; n = 3 independent experiments). C. Effect of Wnt2b and Wnt5a addition on reporter activity. YAMC cells, following transient transfection for 24 h with TOPflash plasmid and with siRNA specific to Wnt2b and Wnt5a, respectively. After 24 hr, cells were incubated with purified Wnt2b or Wnt5a, infected with CR at 90:1 MOI for 3 h, washed to remove bacteria followed by measurement of reporter activity at 48 h using Renilla

Article Snippet: YAMC cells (56105) were transfected with either 100 nmol/L of scrambled siRNA or siRNAs specific for Wnt2b (sc-155356) and Wnt5a (sc-41113) using 2–8 ml of transfection reagent (sc-29528; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 24 h. CR strain DBS 100 (ATCC cat.# 51459TM) was grown under aerophilic conditions on Luria-Bertani (LB) agar plates for 24 h at 37uC and cultured in LB broth O/N at 37uC.

Techniques: In Vitro, Expressing, Infection, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Purification

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: HCC-TCM promotes M2 polarization via Wnt2b/β-catenin signalling. a THP-1 derived macrophages (THP-1-M) were incubated with 50% HCC-TCM for 48 h to obtain the HCC-educated macrophages (HCC-TAMs). Transcript expression levels of Wnt2b were determined in HCC-TAMs by qPCR. b The expression levels of Wnt2b (red) in CD68 + macrophages (green) were determined by immunofluorescence using TMA containing pairs of tumors and matched para-carcinoma tissues of HCC patients. c , d THP-1-M were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry and qPCR, respectively. THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vector were acquired as described in the Materials and Methods, and then incubated with 50% HCC-TCM for 48 h. The expression levels of CD163 and markers for M1 or M2 macrophages on/in these cells were determined by flow cytometry ( e , i ) and qPCR ( f , j ), respectively. The expression levels of β-catenin in HCC-TAMs that were infected with control vectors or sh-Wnt2b vectors were determined by western blotting and immunofluorescence respectively ( g , h ). One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721; TMA, Tissue microarray. Data are presented as mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Active Wnt2B-V5 encoding homo Wnt2b (plasmid #43808; Addgene) was a gift from Xi He (Professor of Neurology, Harvard Medical School, USA).

Techniques: Derivative Assay, Incubation, Expressing, Immunofluorescence, Transfection, Control, Flow Cytometry, Infection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: The activation of Wnt2b/β-catenin signalling enhances TAMs-induced tumour-promoting effects. a THP-1 derived macrophages (THP-1-M) were transfected with control vectors or Wnt2B-V5 (over-Wnt2b) vectors for 48 h. These macrophages were incubated with RPMI 1640 for an additional 24 h to obtain the condition medium (CM). HCC cells were cultured in the presence of indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. ( b-e ) THP-1-M infected with control vectors, sh-Wnt2b or sh-CTNNB1 (β-catenin) vectors were acquired as described in Materials and Methods. These macrophages were incubated with 50% HCC-TCM for 48 h for the preparation of the different TAMs. These TAMs were incubated with RPMI 1640 for another 24 h to obtain the CM. b , c HCC cells were cultured in the presence of the indicated CM for 48 h. The expression levels of EMT markers were determined by western blotting. d HCC cells were incubated with culture medium (Ctrl) or the indicated CM for 24 h. The cell viability of each group was detected by MTT assay. e HCC cells were scratched with a plastic pipette tip and incubated with culture medium (Ctrl) or indicated CM for 24 h. The results of this wound healing assay were photographed and measured. One representative of at least three independent experiments is shown. qPCR, quantitative real-time PCR; HCC, hepatocellular carcinoma; TCM, tumour condition culture medium; TAMs, tumour-associated macrophages; 7721, SMMC-7721. Data are presented as mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Active Wnt2B-V5 encoding homo Wnt2b (plasmid #43808; Addgene) was a gift from Xi He (Professor of Neurology, Harvard Medical School, USA).

Techniques: Activation Assay, Derivative Assay, Transfection, Control, Incubation, Cell Culture, Expressing, Western Blot, Infection, MTT Assay, Transferring, Wound Healing Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: The inhibition of Wnt2b/β-catenin signaling reduces the tumour-promoting effect of HCC-TAMs in vivo. SMMC-7721 cells (6 × 10 6 ) with or without the indicated TAMs (1.5 × 10 6 ) were mixed with Matrigel (at ratio 4:1) and subcutaneously injected into the right subaxillary of 6-week old immunodeficient mice ( n = 6 mice/group). a , d Representative images of the subcutaneous tumors from each group. b , e Growth of subcutaneous tumours (left); the average tumour weight of each group at the time of euthanisation(right). c , f Representative images of immunohistochemistry staining of vimentin, E-cadherin, β-catenin, c-Myc in tumour tissues. g Overall survival HCC patients related to indicated gene expression levels, were generated by the GEPIA2 database via Kaplan–Meier analysis. HCC, hepatocellular carcinoma; TAMs, tumour-associated macrophages; IHC, Immunohistochemistry. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Active Wnt2B-V5 encoding homo Wnt2b (plasmid #43808; Addgene) was a gift from Xi He (Professor of Neurology, Harvard Medical School, USA).

Techniques: Inhibition, In Vivo, Injection, Immunohistochemistry, Staining, Gene Expression, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

doi: 10.1186/s13046-020-01808-3

Figure Lengend Snippet: Schematic representation illustrates the positive feedback loop between HCC cells and HCC-TAMs. Polarization-promoting factors (IL-10, TGF-β, ect.) in the HCC TME can up-regulate the expression of Wnt2b in macrophages, then promote expression and nuclear translocation of β-catenin, which can promote the M2 polarization of TAMs, a process associated with the activation of HCC-TAMs glycolysis by activating c-Myc. These polarized TAMs can promote the proliferation, migration and EMT of tumor cells. TLR9 agonist CpG ODG can act as a blocker of Wnt2b signal which can inhibit M2 polarization of HCC-TAMs induced by HCC-TCM. HCC, hepatocellular carcinoma; TME, tumor microenvironment; TAMs, tumour-associated macrophages; EMT, epithelial-mesenchymal transformation

Article Snippet: Active Wnt2B-V5 encoding homo Wnt2b (plasmid #43808; Addgene) was a gift from Xi He (Professor of Neurology, Harvard Medical School, USA).

Techniques: Expressing, Translocation Assay, Activation Assay, Migration, Transformation Assay