wnt5a expression  (R&D Systems)

Journal: Molecular Cancer

Article Title: Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells

doi: 10.1186/1476-4598-8-90

Figure Lengend Snippet: Wnt5a inhibits canonical Wnt signaling activity in Huh7 and HepG2 cells . ( a ) Huh7 cells were co-transfected with either pCI-neo-mutant β-catenin (S33Y) plasmid (S33Y-β-catenin +) along with pShuttle-IRES-WNT5a or empty pShuttle-IRES vector. 48 hours post transfection; cells were subjected to TCF reporter assay. TCF activity denotes the ratio of signals detected with pGL3-OT (OT) and pGL3-OF (OF) plasmids, respectively. Assays in triplicate, error bars; SD. Co-transfections included pGL-OT or pGL-OF, in addition to pCI-neo-S33Y-β-catenin and pIRES plasmids. ( b ) HepG2 experiments were performed under similar conditions, except that pCI-neo-mutant β-catenin (S33Y) plasmid was omitted.

Article Snippet: The pShuttle-IRES-Wnt5a expression plasmid was constructed by subcloning of an EcoRI-cut Wnt5a cDNA fragment from plasmid pGEMTz-Wnt5a vector (a gift from R. Kemler) into BglII site of the pShuttle-IRES-hrGFP-1 vector (Stratagene, USA). pCI-Neo-mutant β-catenin (S33Y) expression plasmid, and pGL3-OT and pGL3-OF reporter plasmids were kindly provided by B. Vogelstein.

Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Reporter Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: A nerve conduit filled with Wnt5a‐loaded fibrin hydrogels promotes peripheral nerve regeneration

doi: 10.1111/cns.13752

Figure Lengend Snippet: Characterization of the Wnt5a‐loaded fibrin hydrogel. (A–B) Gross view of the saline solution (left) and fibrin hydrogel (right) at upright and upside‐down position. (C) A representative photograph of the nerve conduit filled with the Wnt5a‐loaded fibrin hydrogel. (D) Representative SEM images. (E) The absorbance of Wnt5a when the mass ratio (w/w) of Wnt5a and fibrin hydrogel was 1.2, 1.4, 1.6, and 1.8, respectively. (F) The loading capacity (w/w%) of Wnt5a‐loaded fibrin hydrogel in each group. (G) The sustained‐release profile of Wnt5a‐loaded fibrin hydrogel

Article Snippet: We mixed 100 UI/mL thrombin (T4648, Sigma‐Aldrich) and 0.8% fibrinogen (f8630, Sigma‐Aldrich, T4648, Sigma‐Aldrich) solutions at a ratio of 1:4 to form the fibrin hydrogel., We created a Wnt5a‐loaded fibrin hydrogel by mixing the Wnt5a solution (7815‐NG, R&D Systems) in thrombin and fibrinogen solutions, respectively, before the fibrin hydrogel was synthesized.

Techniques: Saline

Journal: CNS Neuroscience & Therapeutics

Article Title: A nerve conduit filled with Wnt5a‐loaded fibrin hydrogels promotes peripheral nerve regeneration

doi: 10.1111/cns.13752

Figure Lengend Snippet: Wnt5a regulates SC proliferation and secretion. (A–C) qRT‐PCR results showed relative expression levels of VEGF, NGF, and CNTF mRNA. (D–F) ELISA results indicated the concentrations of VEGF, NGF, and CNTF. (G) CCK‐8 results showed the effect of Wnt5a on SC proliferation after incubation for 24 h and 48 h.* p < 0.05, ** p < 0.01, *** p < 0.001. Data are expressed as the mean ±SD. SCs, Schwann cells; VEGF, vascular endothelial growth factor; NGF, nerve growth factor; CNTF, cholinergic neurotrophic factor. qRT‐PCR, quantitative real‐time polymerase chain reaction

Article Snippet: We mixed 100 UI/mL thrombin (T4648, Sigma‐Aldrich) and 0.8% fibrinogen (f8630, Sigma‐Aldrich, T4648, Sigma‐Aldrich) solutions at a ratio of 1:4 to form the fibrin hydrogel., We created a Wnt5a‐loaded fibrin hydrogel by mixing the Wnt5a solution (7815‐NG, R&D Systems) in thrombin and fibrinogen solutions, respectively, before the fibrin hydrogel was synthesized.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Incubation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: Overexpression of Wnt5a in ATL cells. ( A ) The WNT5A mRNA levels were examined in various cell lines. Compared with HTLV-1 uninfected cell lines (Jurkat, CEM, and Molt-4), HTLV-1 infected cell lines (MT-2 and C91/PL) and ATL patient-derived cell lines (HUT102, MT-1, and TL-Om1) show 10 to 100-folds higher WNT5A mRNA levels (left-hand side graph). A similar tendency was also observed at the Wnt5a protein level. Especially, TL-Om1 showed the highest mRNA and protein expression levels of the WNT5A gene. Please note that the right-hand side panel consists of two pictures from two separated blots for Wnt5a and β-Actin, respectively, which are clearly separated by black frames. ( B ) The total WNT5A mRNA levels were re-analyzed in gene expression profiling analysis (left-hand side graph, GSE33615, PBMCs from ATL patients n = 52 and normal CD4 + T cells n = 21, * P < 0.05; *** P < 0.001) and were confirmed in the quantitative-PCR analysis (right-hand side graph, PBMCs from ATL patients n = 5 and normal CD4 + T cells n = 6). Both data demonstrated that the WNT5A mRNA level increased up to 100-folds in PBMCs from ATL patients compared with normal CD4 + T cells. In gene expression profiling data, the WNT5A mRNA level was significantly higher in PBMCs from acute-type ATL patients than indolent-type ATL patients. ( C ) The total WNT5A mRNA levels were examined in primary malignant ATL cells in gene expression profiling analysis (left-hand side graph, GSE55851, Indolent-type ATL n = 5 and acute-type ATL n = 3, *** P < 0.001) and in quantitative-PCR analysis (right-hand side graph, asymptomatic carrier n = 1, indolent-type ATL n = 1, and acute-type ATL n = 3). In both experiments, malignant ATL cells were isolated as CADM1 + /CD7 − CD4 + T cells . Both graphs show that the expression level of the total WNT5A mRNA increases about 100-folds in acute-type cells compared with that in indolent-type ATL cells.

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Over Expression, Infection, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Isolation

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: Wnt5a dependent NF-κB activation and cell growth. ( A ) IWP-2 treatment in Jurkat, CEM, Molt-4, MT-2, HUT102, and TL-Om1 cells. The dose-dependent suppression of cell growth was observed in MT-2 and TL-Om1, which expressed high levels of Wn5a protein (the upper panel). Please note that the upper panel consists of two pictures from two separated blots for Wnt5a and β-Actin, respectively, which are clearly separated by black frames. ( B ) IWP-2 treatment significantly and dose-dependently suppressed cellular NF-κB activities in Jurkat and TL-Om1 cells (n = 6, mean ± SD, * P < 0.05). ( C ) Specific knockdown of Wnt5a expression (the upper panel) by shRNA against WNT5A mRNA in Jurkat, MT-2, and TL-Om1 significantly reduced the cellular survival rates. Please note that the upper panel consists of two pictures from two separated blots for Wnt5a and β-Actin, respectively, which are clearly separated by black frames. The graphs show the course of changes in the percentage of Venus (+) cells, i.e., Wnt5a knockdown cells, after the recombinant lentivirus infection. The % Venus (+) cells in sh WNT5A expressing cells decline rapidly during infection, while that with sh LUC (the negative control) does not. The induction of cell-death in Wnt5a knockdown cells were more significant in MT-2 and TL-Om1 cells than Jurkat, in agreement with IWP-2 treatments in ( A ).

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Activation Assay, Expressing, shRNA, Recombinant, Infection, Negative Control

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: c-Myb and FoxM1 synergistically upregulate the WNT5A gene expression in ATL cells. ( A ) Re-analysis of the gene expression microarray data (GSE33615) show that FOXM1 and MYB mRNA levels increase in ATL primary PBMCs compared with normal CD4 + T cells (PBMCs from ATL patients n = 52 and normal CD4 + T cells n = 21, * P < 0.05; ** P < 0.01). ( B ) WNT5A , FOXM1 , and MYB mRNA levels are increased in correlation in PBMCs from ATL patients (GSE33615, PBMCs from ATL patients n = 52 and normal CD4 + T cells n = 21). In the graphs, the x-axis shows FOXM1 mRNA levels, the y-axis shows MYB mRNA levels, and the size of the bubble indicates WNT5A mRNA levels. Those three mRNA levels are low in Normal CD4 + T cells (the left-hand side graph), while they are increased altogether in ATL cells (the right-hand side panel). ( C ) The molecular relationship among Wnt5a, FoxM1, and c-Myb was investigated by ChIP assays and luciferase-based promoter activity assays (n = 6, mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001). c-Myb directly binds to the FOXM1 promoter region and transactivates the FOXM1 gene (the top-left), and vice versa (the top-right). Both c-Myb and FoxM1 are recruited to the promoter region of the WNT5A gene, and transactivate the WNT5A promoter (the lower graphs). The c-Myb-9A isoform, which lacks the C-terminal negative regulatory domain and shows significantly higher transactivity than the WT-c-Myb, demonstrated significantly higher recruitment and activation of FOXM1 promoter and WNT5A promoter than WT-c-Myb (the top-left and the lower-left). ( D ) Simultaneous expression of FoxM1 and c-Myb elevated WNT5A promoter activity in a synergistic manner compared with the cases in that FoxM1 or c-Myb was expressed alone. Moreover, co-expression of the c-Myb-9A and FoxM1 showed more than twofold higher WNT5A transactivity compared with co-expression of the WT-c-Myb and FoxM1 (n = 6, mean ± SD, ** P < 0.01; *** P < 0.001).

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Expressing, Microarray, Luciferase, Activity Assay, Activation Assay

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: The ΔExon4-WNT5A transcript variant and the ΔC-Wnt5a are frequently overexpressed in acute-type ATL cells. ( A ) The semi-quantitative RT-PCR for the WNT5A mRNA in total RNA samples extracted from normal CD4 + T cells (n = 6), HTLV-1 asymptomatic carriers (ACs; n = 6), and ATL patients (smoldering type; n = 2, chronic type; n = 10, and acute type; n = 17). We designed the primers at the exon3 and exon5 of human WNT5A mRNA, which amplifies a 630 bp fragment for the WT-WNT5A mRNA, while a 293 bp fragment for the WNT5A mRNA without the exon4 (the ΔE4-WNT5A mRNA). The left-hand side panel shows the representative data. The rest of the data are shown in Figure S1. Please note that the left-hand side panel consists of two pictures from two separated agarose gels for WNT5A mRNA and GAPDH mRNA, respectively, which are clearly separated by black frames. Semi-quantitative RT-PCR with these primers showed that the frequency of the ΔE4-WNT5A mRNA reached 76% in acute type ATL patients, followed by 60% in chronic type ATL patients (the right-hand side table). ( B ) The ΔE4-WNT5A mRNA encodes a C-terminal truncated short Wnt5a isoform of 136 amino acids (the upper panel). The lower panel is the Western blotting with anti-Wnt5a antibody against the N-terminus epitope. The results show the bands of the ΔC-Wnt5a at 15 kDa are detected only in acute-type ATL samples (the right panel). The left panel shows the Western blotting of the WT-Wnt5a-His and the ΔC-Wnt5a-His overexpressed in HEK293FT cells as the reference. Please note that both the left- and the right-hand side panels consist of two pictures from separated blots for Wnt5a and β-Actin, respectively, which are clearly separated by black frames.

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Variant Assay, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: The ΔC-Wnt5a is secreted via a non-canonical secretion pathway. ( A ) In order to test if the ΔC-Wnt5a is secreted out of the cell, we design an assay using Wnt5a fused with NanoLuc at C-terminal (the upper panel). The NanoLuc assays were conducted in the whole cell lysate (L) and the culture medium (M) of HEK293FT cells overexpressing Wnt5a-NanoLuc (the lower left-and side graph, n = 4, mean ± SD, *** P < 0.001), and the secretion rate was calculated as (NanoLuc signal in medium/NanoLuc signal in cell lysate) (the lower right-hand side graph, n = 4, mean ± SD, *** P < 0.001). The secretion rate was about 4-times higher in the ΔC-Wnt5a compared with the WT-Wnt5a. ( B ) IWP-2 inhibits the secretion of Wnt family proteins, including Wnt5a. IWP-2 treatments in MT-2 cells reduced the Wnt5a level detected in the culture medium in a dose-dependent manner, which confirmed that IWP-2 indeed inhibited the secretion of Wnt5a in MT-2 cells (the left-hand side panel). Please note that the left-hand side panel consists of three pictures from three separated blots for the WT-Wnt5a and β-Actin in cell lysate and the WT-Wnt5a in culture medium, respectively, which are clearly separated by black frames. The right-hand side graph shows the secretion rates of the WT-Wnt5a and the ΔC-Wnt5a overexpressed in HEK293FT cells, which were treated with IWP-2 of indicated doses (n = 4, Mean ± SD, *** P < 0.001). The IWP-2 treatment reduced the secretion rate of the WT-Wnt5a significantly, while it did not alter the secretion rate of the ΔC-Wnt5a.

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques:

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: The ΔC-Wnt5a induces a higher cell velocity and an invasion rate than the WT-Wnt5a. ( A ) HEK293FT cells overexpressing Wnt5a-His and Lifeact-GFP were subjected to the cell velocity analysis. The movements of HEK293FT cells (expressing mock, the WT-Wnt5a-His, or the ΔC-Wnt5a-His, 10 cells each) were monitored with BioStation live cell imaging system (Nikon, Corp.), and the mean velocity was calculated using ADAPT software. The analysis shows that the mean velocity significantly increased in the ΔC-Wnt5a overexpressing cells compared with the mock cells with the empty vector (n = 10, mean ± SD, * P < 0.05). ( B ) Wound-healing assays were conducted in HEK293FT cells overexpressing the empty vector (mock), the WT-Wnt5a-His, or the ΔC-Wnt5a-His. The wound-healing rate was calculated at 24, 48, and 72 h after the wound formation as {100% − [(wound area at the time)/(wound area at 0 h) × 100%)]}. The graph demonstrates that Wnt5a expressing cells show significantly higher wound-healing rates compared with the mock control cells. Moreover, the wound-healing rate of the ΔC-Wnt5a expressing cells was significantly higher than the WT-Wnt5a expressing cells at 24 h (n = 6, mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Expressing, Live Cell Imaging, Software, Plasmid Preparation

Journal: Scientific Reports

Article Title: Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

doi: 10.1038/s41598-021-83613-2

Figure Lengend Snippet: The ΔC-Wnt5a induces strong chemotaxis to CXCL12 and support cell growth. ( A ) The upper panels show the expression of CXCR4 in Jurkat, CEM, TL-Om1, and MT-1 cells measured by flowcytometry. The lower graphs show the relative migration rates toward CXCL12 in Jurkat, CEM, TL-Om1, and MT-1 cells incubated in the medium containing the WT-Wnt5a or the ΔC-Wnt5a (n = 6, mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001). Recombinant human Wnt5a (rWnt5a) was added at 100 ng/mL to the medium as a positive control, which induced significantly higher migration rates compared with the negative controls (medium only) in all tested cell lines. The WT-Wnt5a did not stimulate the cellular chemotaxis to CXCL12 except for MT-1. The ΔC-Wnt5a significantly enhanced the chemotaxis to CXCL12 in all cell lines to the same extent compared with the rWnt5a. The treatment with a CXCR4 antagonist (CXCR4i) abolished the cellular migration to CXCL12 in all cell lines. Thus, CXCL12/CXCR4 chemotaxis played a major role in the cellular migration observed in these assays. ( B ) The cell viability assays were conducted in Jurkat and TL-Om1 cells cultured in the medium containing the WT-Wnt5a or the ΔC-Wnt5a. The relative viability was significantly elevated in the both cell lines, which were cultured in the medium containing the ΔC-Wnt5a compared with the cells cultured in mock medium (n = 6, mean ± SD, * P < 0.05; *** P < 0.001). The relative cell viability was significantly elevated only in TL-Om1 cells, which were cultured in the medium containing the WT-Wnt5a (n = 6, mean ± SD, * P < 0.05). ( C ) Summary of this study. We demonstrated that both FoxM1 and c-Myb transactivate Wnt5a expression. Since FoxM1 and c-Myb transactivate each other, elevated levels of FoxM1 and c-Myb in ATL cells synergistically enhance the Wnt5a expression. Such feed-forward molecular mechanism may cause the drastic overexpression of Wnt5a in acute-type ATL cells. Moreover, in acute-type ATL cells, the Wnt5a level is not only elevated, but also a mutant, yet functional Wnt5a, the ΔC-Wnt5a, is overexpressed. Overexpression of the ΔC-Wnt5a may accelerate the dysregulation in the Wnt5a pathway, which may be responsible for malignant phenotypes of acute-type ATL cells, such as uncontrollable cell proliferation, migration, and invasion.

Article Snippet: For further investigation of the extracellular secretion characteristics of the ΔC-Wnt5a, the WT- and the ΔC-Wnt5a expression plasmids with NanoLuc (Promega, Corp.) at the C-terminus were prepared (Fig. A, upper panel).

Techniques: Chemotaxis Assay, Expressing, Migration, Incubation, Recombinant, Positive Control, Cell Culture, Over Expression, Mutagenesis, Functional Assay