Journal: Advanced Science

Article Title: Aberrant Cholesterol Metabolism and Wnt/ β ‐Catenin Signaling Coalesce via Frizzled5 in Supporting Cancer Growth

doi: 10.1002/advs.202200750

Figure Lengend Snippet:

Article Snippet: L‐Wnt3a cell , ATCC , CRL‐2647.

Techniques: Staining, Western Blot, Sequencing, Control

Journal: International Journal of Nanomedicine

Article Title: Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy

doi: 10.2147/ijn.s23747

Figure Lengend Snippet: Figure 5 Bcl-2, Cyclin D1, and Wnt3a knock down following miRNA/PAMAM-PEG-APT complexes delivery to LNCaP cells. The cells were treated with miRNA/ PAMAM-PEG-APT (miRNA) and NC-miRNA/PAMAM-PEG-APT (NC-miRNA) complexes and processed for immunoblotting with anti-Bcl-2, anti-Cyclin D1, and anti-Wnt3a antibodies at 24, 48, and 72 hours post-transfection. β-actin was a loading control. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Article Snippet: The blotted membrane was immunostained with antibodies specific to anti-Bcl-2 (BioWorld, Visalia, CA), anti-Cyclin D1 (BioWorld) and anti-Wnt3a antibodies (R&D Systems Inc, Minneapolis, MN).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Cancer Research Communications

Article Title: WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers

doi: 10.1158/2767-9764.CRC-23-0275

Figure Lengend Snippet: BioID supports WNT4 localization to the mitochondria. A, Proteins enriched in HT1080 Wnt-BirA versus parental HT1080 cells lacking BirA construct expression. B, Overlap of proteins identified in HT1080 (A) versus HT1080-PKO and MM134 identifies n = 72 “high-confidence” WNT4-associated proteins. C, Gene ontology analysis for cellular compartment for WNT3A- versus WNT4-associated proteins. Dashed line = 1.3 ( P = 0.05). D, Network analysis of WNT3A- versus WNT4-associated proteins via subcell barcode. Enrichments against cell line HCC287 background shown; parallel results observed with other cell line background data, for example, MCF7. E, Proteins with predicted cytosolic or mitochondrial localization (subcell barcode) among “high-confidence” WNT4-associated proteins. Red = predicted mitochondrial localization, pink = mTOR complex in mitochondrial dynamics, biogenesis, and autophagy. F, Biotin treatment and streptavidin pulldown was performed as for MS studies, and candidate WNT4-associated proteins from E detected by immunoblotting. Total protein by Ponceau.

Article Snippet: Blots were probed with Streptavidin-HRP (Cell Signaling Technology #3999; RRID:AB_10830897) or antibodies used according to manufacturer's recommendations: WNT4 (R&D Systems, MAB4751; RRID:AB_2215448); WNT3A (Novus Biologicals, MAB13242); anti-HA-HRP conjugate (Cell Signaling Technology #2999; RRID:AB_1264166); DHRS2 (Sigma HPA053915; RRID:AB_2682307); mTOR (Cell Signaling Technology #2983; RRID:AB_2105622); STAT1 (Sigma HPA000931; RRID:AB_1080100).

Techniques: Construct, Expressing, Western Blot

Journal: eLife

Article Title: Anti-resonance in developmental signaling regulates cell fate decisions

doi: 10.7554/eLife.107794

Figure Lengend Snippet: ( A ) FACS data for Wnt I/O cell line with (+)/without (-) 24 hr of light exposure. Axes are of β-catenin (mCherry) v. TopFlash (APC-Cy5.5) ( B ) Live cell imaging of CRISPR tdmRuby3-β-cat, lentiviral 8X-TOPFlash-tdIRFP and DAPI, 16 hr after adding CHIR99021 (+CHIR) and Wnt3a (+Wnt3 a). ( C ) Quantifications of β-catenin and TopFlash fluorescence for no light, +16 hr light, +Wnt3 a, and +CHIR, where the mean of each condition was normalized to the mean of the no light condition and error bars represent SEM. ( D ) Flow for cell segmentation and heatmap generation from experimental data, using CellPose add TrackMate. From left to right: magenta (β-catenin) and cyan (TopFlash) images are passed into CellPose + Trackmate for segmentation and tracking. An example image of CellPose segmentation is shown under ‘segmented images,’ where the different colors correspond to the cell’s segmentation ID. Final images under ‘quantify and plot’ show quantification of β-catenin and TopFlash. ( E ) Mean fluorescent intensity (MFI) of β-catenin in the 24 hr light on condition, normalized to light off β-catenin. ( F ) MFI of TopFlash in the 24 hr light on condition, normalized to light off TopFlash. ( G ) Population mean MFI of β-catenin from live, single cell traces in indicated conditions, normalized to light off β-catenin. Initial drop in fluorescence is due to media bleaching. ( H ) Population mean MFI of TopFlash from live, single cell traces from indicated conditions, normalized to light off TopFlash. ( I ) Jitter plot of β-catenin mean nuclear fluorescent intensity (MFI) at the maximum intensity point in continuous light exposure conditions. Each point represents a single cell and the black line represents the mean of the population. ( J ) Jitter plot of TopFlash mean nuclear fluorescent intensity (MFI). Each point represents a single cell and the black line represents the mean of the population.

Article Snippet: HEK293T cells were treated using 10 μM of CHIR99201 (Stem Cell Technologies, 72052) or 2.5 nM of Wnt3a ligand (R&D Systems, 5036-WN) added into Dulbecco’s Modified Eagle Medium, high glucose GlutaMAX (Thermo Fisher Scientific, 10566016) medium supplemented with 10% fetal bovine serum (Atlas Biologicals, F-0500-D) and 1% penicillin-streptomycin.

Techniques: Live Cell Imaging, CRISPR, Fluorescence

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: 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