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