Overview of Experimental Design


 

Procedural Overview

O-GlcNAc sites at the N-Terminal (NT) of β-catenin were determined using Green Fluorescent Protein (pEGFP) tagged β-catenin constructs.  A previous study has reported that Plakloglobin, a homologous protein to β-catenin, was O-GlcNAc modified at Threonine 14. Through sequence alignment, this site was proven to be Serine 23 of β-catenin.
 

Cultured prostate cancer (PCa) DU145 cells were transfected with pEGFP-tagged Wild Type (WT) or GFP-tagged β-catenin mutated at Serine 23 to a Glycine (S23G) plasmid constructs already made available. pEGFP empty vector (constructs with no β-catenin sequence) served as the control. Transfected cells were treated with PUGNAc to increase the global levels of O-GlcNAcylation. PUGNAc is an analogue of the O-GlcNAc moiety which competitively binds to O-GlcNAcase. Cells were housed in a temperature controlled incubator: 37 Degrees Celcius, 5% CO2.

Upon PUGNAc treatment, endogenous β-catenin and fusion proteins were immunoprecipitated from whole cell or nuclear lysates with anti-β-catenin and anti-GFP antibodies.  Alterations in β-catenin’s interactions with E-cadherin or TCF (its major cellular interactors) were detected by anti-E-cadherin or anti-TCF antibodies and characterized via Western Blot (WB) analysis. Induction of O-GlcNAcylation was verified either by precipitation with Wheat Germ Agglutinin (WGA) agarose beads or WGA-Horse Radish Peroxidase (HRP). WGA is a lectin that specifically detects O-GlcNAc groups.

The subcellular localization of β-catenin was characterized by immunofluoresence analysis. Endogenous β-catenin was visualized with anti-β-catenin antibody, followed by Alexafluor 488 tagged secondary antibody. WT and S23G β-catenin were followed by their pEGFP tag. Alterations in transcriptional activity of WT and S23G β-catenin with or without PUGNAc treatment were verified by a reporter gene luciferase assay: TOP/FOPflash. The TOP reporter construct is specific for β-catenin transcriptional activity, comprising 3 tandem repeats of TCF binding sites. FOP is the negative control with a mutated TCF binding site. pRenilla served as the transfection control. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) was performed to verify corresponding changes in β-catenin’s target gene expression, which included Cyclin D1 and Vascular Endothelial Growth Factor (VEGF).

To determine the mechanism by which β-catenin is O-GlcNAc modified, DU145 PCa cells were treated with either recombinant Wnt3a or an inhibitor of PI3K, LY294002, for a set time course. DU145 cell lines were used as these cells have a regulated Wnt and PI3K signaling pathway. Total levels of β-catenin and alterations in the O-GlcNAcylation of β-catenin were determined by anti-β-catenin antibody and immunoprecipitation by WGA-agarose beads, respectively, via Western Blot analysis. Inhibition of the PI3K pathway was determined by decreased levels of phosphorylated AKT.

Parallel experiments were performed in primary and metastatic Osteosarcoma cell lines, U2OS and SAOS2 respectively, to assess the universality of β-catenin O-GlcNAcylation. Cells (not transfected) were treated with PUGNAc and alternation in levels of O-GlcNAcylated β-catenin was characterized by Western Blot Analysis. Immunofluoresence was performed to assess the subcellular distribution of β-catenin within these cell lines.


 
Experimental Protocols
  

Transfection: Introduction of plasmid DNA contructs to DU145 PCa cell line. DU145 PCa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) 10% Fetal Bovine Serum (FBS) 1% Penicillin/Streptomyacin. Cells were transfected with pEGFP empty vector (Control), pEGFP-WT β-catenin (Wild Type), pEGFP-S23G β-catenin (β-catenin mutated at Serine 23 to a Glycine) in OPTIMEM using Lipofectamine2000 as per manufacturer’s protocols. Lipofectamine is a common transfect reagent used to facilitate DNA uptake by forming liposomes. These liposomes entrap plasmids and greatly enhance movement across the phospholipid bilayer. (Note: All figures in this section were either manually drawn or waived of copyright)

 
                                                                    Schematic Representation of a Transfection Procedure

PUGNAc Treatment: Increasing global levels of O-GlcNAcylation. Upon successful transfection of DNA constructs, DU145 cells were treated with 100uM PUGNAc for a set time course. Treated cells were washed with PBS and lysed with NP-40/DOC Lysis Buffer, separated into nuclear and cytosolic fractions using Nuclear-Cytosolic Fractionation protocols, or harvested for RNA extraction.

Schematic Representation of PUGNAC Treatment

Immunoprecipitation: Determination and evaluation of O-GlcNAcylation and protein-protein interactions of β-catenin. Total protein concentrations of whole cell or nuclear lysates were determined by Bicinchoninic Acid (BCA) Protein Determination Assay. 100ug of protein were precipitated or immunoprecipitated with WGA-Agarose beads or anti-GFP antibody, respectively. Whole cell and nuclear lysates were incubated with respective antibodies pre-absorbed to Protein A/G beads. Samples were incubated overnight at 4 Degrees Celcius. Total non-specific IgG antibody served as the control. Beads were eluted using SDS PAGE sample buffer. Complexes were separated using Tricine Gel Electrophoresis and characterized by Western Blot analysis. O-GlcNAcylation was determined by WGA-HRP. Alterations in β-catenin and its interactors were analyzed by anti-β-catenin. anti-E-cadherin, and anti-TCF antibodies.

  

Schematic Representation of WGA-agarose Precipitation and Immunoprecipitation Procedure

Western Blot Analysis: Detection and Characterization of Proteins. Total protein concentrations of DU145 PCa cell lysates (whole cell and/or nuclear/cytosolic fractions) were quantified using BCA Protein Determination Assay. Proteins were separated by Tricine Gel Electrophoresis and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes using Towbin's Buffer. PVDF membranes were blocked with skim milk or Bovine Serum Albumin (BSA) for 1 hour at room temperature (or at 4 Degrees Celcius overnight). Primary antibodies were added according to the specifications of the manufacturer's protocols. Corresponding Horse Radish Peroxidase (HRP) conjugated secondary antibodies were added at 1:10 000 dilution for 1 hour at room temperature. Membranes were washed with Tris Buffered Saline + Tween-20 (TBST). Membranes were exposed upon addition of chemiluminescent reagent using X-ray film. Histograms are representative of experiments repeated 3 or more times. Alterations observed by Western Blot analysis were quantified using ImageJ Software (Densitometric Analysis). Statistical analysis was performed using SigmaPlot Software.

Schematic Representation of Western Blot Procedure 

Immunofluorescence Analysis: Visualization of β-catenin’s subcellular localization. DU145 PCa cells were grown on coverslips and subsequently transfected with pEGFP Empty Vector (control), pEGFP-WT-β-catenin, pEGFP-S23G-β-catenin. Cells were then treated 100uM PUGNAc for a set time course. Upon completion of PUGNAc treatment, DU145 cells were washed with PBS and fixed with 4% paraformaldehyde. Cell membranes were permeabilized using ice-cold Methanol. Permeabilized cells were treated with specific primary antibodies and then subsequently followed by fluorescent-tagged secondary antibodies for visualization. pEGFP tagged constructs were followed be observing the GFP signal. Immunofluorescence images were captured with confocal microscope.

Reporter Gene Assay: TOPFlash/FOPflash Luciferase Assay- Quantification of  β-catenin’s Transcriptional Activity. DU145 PCa cells were co-transfected with either TOPflash or FOPflash luciferase construct, and pEGFP empty vector, pEGFP-WT-β-catenin, or pEGPF-S23G-β-catenin constructs. pRenilla luciferase construct served as the transfection control. FOPflash was the negative control. Upon PUGNAc treatment, cells were lysed using Passive Lysis Buffer and the assay was prepared using the Promega Dual Luciferase Assay Kit as per manufacturer's instructions. Luciferase activity was monitored using an available luminometer at the University of Alberta.

Quantative Reverse Transcription Polymerase Chain Reaction: Quantification of mRNA gene expression. Total RNA was isolated using RNeasy Kit as per manufacturer’s protocols. One microgram of total RNA was used for reverse transcription of Oligo (dT) and Superscript III reverse transcriptase. Real-time quantification of Cyclin D1 and Vascular Endothelial Growth Factor (VEGF) was performed using power SYBR Green PCR Master Mix. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. Samples were amplified with a precycling hold at 95 Degrees Celcius for 15 seconds, 30 cycles of annealing and extension at 60 Degrees Celcius for 1 minute.

Recombinant Wnt3a and LY294002 treatment: Determination of the mechanism of O-GlcNAcylation of β-catenin. A) DU145 cells were serum starved for 18 hours then treated with either 150ng/ml or 300ng/ml of Wnt3a for a set time course. Cells were lysed using NP40/DOC lysis buffer. Protein concentrations were quantified using BCA Assay and alterations in O-GlcNAcylation was determined by Western Blot Analysis. Successful Wnt activation was characterized by the stabilization of β-catenin. B) DU145 cells were serum starved for 18 hours and subsequently serum activated with 10% FBS and treated with either 50nM or 100nM LY294002. Cells were lysed using NP40/DOC lysis buffer. Protein concentrations were quantified using BCA Assay and alterations in O-GlcNAcylation was determined by WB analysis. Inhibition of the PI3K pathway was verified with the expected decrease in Phosphorylated (P)-AKT levels.