Journal: Neoplasia (New York, N.Y.)

Article Title: An AXIN2 Mutant Allele Associated With Predisposition to Colorectal Neoplasia Has Context-Dependent Effects on AXIN2 Protein Function 1

doi: 10.1016/j.neo.2015.04.006

Figure Lengend Snippet: AXIN2- and trAXIN2-mediated inhibition of Wnt/β-catenin/TCF transcriptional targets is context dependent. (A) Transient ectopic expression of AXIN2 and trAXIN2 suppress Wnt3a-mediated TCF reporter gene activity. HEK293T cells were transiently transfected with the pCMV-3Tag vector, AXIN2 , or trAXIN2 expression constructs as well as TOPFlash reporter vector. Twenty-four hours after transfection, cells were treated with Wnt3a for 8 hours before harvesting for luciferase assays. Luciferase assays were performed in triplicate and mean and SDs are indicated. (B and C ) Stable expression of AXIN2 but not trAXIN2 inhibits Wnt3a-mediated induction of endogenous Wnt/β-catenin/TCF target genes in rat intestinal IEC-6 cells. IEC-6 cells were transduced with empty retroviral expression vector construct or constructs for AXIN2 or trAXIN2 , and drug selection was undertaken to create stable polyclonal cell lines. IB studies of the resultant IEC-6 cell lines show stable expression of AXIN2 or trAXIN2, as detected with anti-AXIN2 antibody (B). Stable IEC-6 transductants were treated for 16 hours with Wnt3a. The cells were then harvested, total RNA was collected, and expression of the indicated Wnt / β-catenin / TCF target genes was assessed in three separate quantitative RT-PCR experiments. The individual data points for three independent qPCR experiments with the mean of each group designated by a horizontal line are shown in C.

Article Snippet: Twenty-four hours after plating, the cells were treated with 200 ng/ml recombinant Wnt3a (ProSci, San Diego, CA) to induce target gene expression or with phosphate-buffered saline as a control.

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Transduction, Retroviral, Selection, Quantitative RT-PCR

Journal: Journal of neuroinflammation

Article Title: Involvement of Wnt7a in the role of M2c microglia in neural stem cell oligodendrogenesis.

doi: 10.1186/s12974-020-01734-3

Figure Lengend Snippet: Fig. 7 M1 microglia secrete Wnt5a, while M2c microglia secrete the Wnt7a protein. a OX-42 staining of microglial cells following 24 h of stimulation with polarization agents to obtain the M1, M2a, and M2c activation states. b, d Wnt5a and Wnt7a gene expression by microglia in different activation states after 6 and 24 h of polarization. c, e Wnt5a and Wnt7a protein secreted after 24 h of polarization, evaluated in the supernatant of the cultures by ELISA. N = 4 independent experiments with three replicates. Statistics: *p ≤0.05; **p ≤0.01; ***p ≤0.001 vs. Control; ANOVA followed by Bonferroni’s post-hoc test. Scale bar: 25 μm

Article Snippet: The Wnt5a and Wnt7a in the supernatants of microglia cultures were measured using specific solid-phase sandwich ELISA kits (CSB-EL026138RA and CSB-EL026141MO, Cusabio) following the manufacturer’s recommendations (four independent experiments with three replicates were studied).

Techniques: Staining, Activation Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Oncology

Article Title: FUT2 promotes the tumorigenicity and metastasis of colorectal cancer cells via the Wnt/β-catenin pathway

doi: 10.3892/ijo.2023.5483

Figure Lengend Snippet: Effects of FUT2 on Wnt2 in colorectal cancer cells. (A) The expression of Wnt2 in SW-480 cells was evaluated using western blot analysis. (B) The α-1,2-fucosylation of Wnt2 was detected by UEA-1 lectin pull-down assay in SW-480 cells. (C) The interaction between FUT2 and Wnt2 was detected using co-immunoprecipitation assay. FUT2, fucosyltransferase 2.

Article Snippet: Primary antibody against glycogen synthase kinase-3β (GSK3β; cat. no. 12456) was acquired from Cell Signaling Technology, Inc. Primary antibodies against β-catenin (51067-2-AP), and Wnt2 (66656-1-Ig) were obtained from Proteintech Group, Inc. Antibodies against tubulin (db3285), GAPDH (db1209) and β-actin (db10001) were purchased from Diagbio ( http://www.daigebio.com ), and used as the loading controls.

Techniques: Expressing, Western Blot, Pull Down Assay, Co-Immunoprecipitation Assay

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 1. miR‑214 is downregulated in liver cancer and targets Wnt3a. (A) Reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression of miR‑214 in 24 paired human hepatocellular carcinoma and non‑tumor tissues. (B) Relative expression of miR‑214 in liver cancer cell lines and a normal liver cell line. **P<0.01; *P<0.05. (C) miR‑214 seed region sequence in the 3'UTR of Wnt3a. (D) Wnt3a protein expression as detected by immunohistochemistry. (E) Protein expression levels of Wnt3a were measured by western blot analysis in HepG2 cells transfected with miR‑214 or miR‑ctrl. (F) miR‑214 was co‑transfected with pmirGLO, pmirGLO‑Wnt3a‑3'‑UTR‑wt or pmirGLO‑Wnt3a‑3'‑UTR‑mut in HepG2 cells. Relative luciferase activity was measured after 48 h. *P<0.05 vs. control. miR, microRNA; mut/M, mutant; UTR, untranslated region; wt/W, wild‑type.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Polymerase Chain Reaction, Expressing, Sequencing, Immunohistochemistry, Western Blot, Transfection, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 2. miR‑214 inhibits the proliferation of liver cancer cells. CCK8 assay was performed to detect the effects of miR‑214 on cell proliferation at 24, 48, and 72 h in (A) HepG2 and (B) Hep3B cells. CCK8 assay was performed to detect the effects of siWnt3a on cell proliferation at 24, 48 and 72 h in (C) HepG2 and (D) Hep3B cells. Wnt3a overexpression vector was co‑transfected with miR‑ctrl or miR‑214 into (E) HepG2 and (F) Hep3B cells, and cell proliferation was detected by CCK8 assay. *P<0.05; **P<0.01 vs. miR‑ctrl + Wnt3a‑ctrl. CCK8, Cell Counting kit‑8; ctrl, control; miR, microRNA; OD, optical density; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 3. Overexpression of miR‑214 or Wnt3a silencing affects cell cycle progression. Cell cycle analysis of (A) HepG2 and (B) Hep3B cells following transfection with miR‑214 or miR‑ctrl for 48 h. Cell cycle analysis of (C) HepG2 and (D) Hep3B cells following transfection with siWnt3a or si‑ctrl for 48 h. *P<0.05. ctrl, control; miR, microRNA; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Over Expression, Cell Cycle Assay, Transfection, Control, Small Interfering RNA

Journal: PeerJ

Article Title: Platelet factor 4 inhibits human hair follicle growth and promotes androgen receptor expression in human dermal papilla cells

doi: 10.7717/peerj.9867

Figure Lengend Snippet: (A–H) Effects of PF4 on hair growth-promoting and DP signature genes ( Wnt5a, Wnt10b , DKK1 , LEF1, HEY1, IGF-1, BMP2 and BMP4 ) mRNA expression in human DPCs cultured for 24 h. (I–K) Effects of PF4 on protein expression of Wnt5a, IGF-1 and BMP2 in human DPCs cultured for 48 h. Data are reported as mean + SEM. Student’s t-test was used to compare data. * P < 0.05, ** P < 0.01. “ns” indicates no significant difference.

Article Snippet: Wnt5a, IGF1 and BMP2 were measured by Wnt5a, IGF1 and BMP2 ELISA kits (CUSABIO) following the manufacturer’s instruction, respectively.

Techniques: Expressing, Cell Culture