Experimental Data

NOTE: This study was carried out under the supervision of Dr. Sujata Persad at the University of Alberta. All experimental data are thus the property of the University of Alberta, Persad Laboratory. Distribution is strictly prohibited.


 

Figure 1 – PUGNAc increases O-GlcNAcylation of endogenous β-catenin. DU145 cells were treated with PUGNAc (100 µM) and whole cell lysates were immunoprecipitated for β-catenin. Upon PUGNAc treatment, levels of O-GlcNAc modified β-catenin significantly increased. Total cellular levels of β-catenin remained stable. The absence of any signal for the IgG control indicates the absence of non-specific binding of antibodies used. Densitometric analysis was performed on 3 or more independent experiments with the control condition set at 100%.*p<0.001.


 

Figure 2 – Serine 23 is a site for O-GlcNAcylation. DU145 cells transfected with pEGFP tagged WT or mutant S23G β-catenin treated with (±) PUGNAc. Levels of O-GlcNAc of full length WT β-catenin increased with PUGNAc. O-GlcNAc levels of mutant S23G β-catenin was minimal despite PUGNAc treatment. *p<0.001.

 
 
Figure 3 – O-GlcNAcylation affects β-catenin’s subcellular localization. Immunofluorescence analysis of the subcellular localization of endogenous, WT, and Mutant S23G β-catenin. Panel 1) Endogenous β-catenin  is widely dispersed throughout the cytoplasm and the nucleus. Upon PUGNAc treatment, β-catenin localized to the plasma membrane, characteristic of the stable epithelial phenotype. The pEGFP-WT-β-catenin construct behaved similarly (Panel 2). PUGNAc had no effect on the S23G Mutant construct (Panel 3). Nucleus-Blue, was stained with DAPI.  Endogenous β-catenin was visualized using Alexafluor 488 secondary antibody to β-catenin primary antibody. WT and Mutant β-catenin constructs were followed using their pEGFP tag.


Figure 4 - O-GlcNAcylation increases β-catenin's interaction with E-cadherin while S23 mutation minimizes this interaction. 
A) Immunoprecipitation of PUGNAc treated DU145 lysate resulted in increased binding of endogenous β-catenin with E-cadherin. B) Immunoprecipitation of PUGNAc treated DU145 lysate resulted in increased binding of WT β-catenin with E-cadherin while there was no change in interactions observed with the S23G mutant β-catenin. * p<0.001.

Figure 5 – O-GlcNAcylation decreases β-catenin's nuclear localization and its interaction with TCF; S23 mutation results in maximal TCF interaction. A) Nuclear lysates of DU145 cells transfected with WT or S23G mutant full length β-catenin were characterized by Western Blot Analysis. Levels of WT-β-catenin decreased within the nucleus upon PUGNAc treatment. No change was observed in S23G mutant β-catenin. Lamin B characterizes an enriched nuclear fraction.The absence of alpha-beta tubulin indicates a nuclear fraction free from cytosolic contamination. Cytosolic fractions were immunoprecipitated for β-catenin to ensure presence of O-GlcNAcylation. B) Immunoprecipitation of PUGNAc treated DU145 nuclear lysate showed significantly decreased binding of WT β-catenin with TCF while S23G mutant β-catenin exhibited no change. *p<0.001.


 
 
 
Figure 6 – O-GlcNAcylation of β-catenin decreases Wnt target gene expression while S23 mutation maximizes target gene expression. RNA was extracted from DU145 cells transfected with WT or S23G mutant β-catenin and treated with or without PUGNAc. pEGFP empty vector served as the control. RT-qPCR was performed on 1ug of RNA extract. mRNA copy number of A) Cyclin D1 and B) VEGF decreased for WT and control conditions upon PUGNAc treatment. S23G mutant β-catenin transfected cells showed no change in mRNA copy number of Cyclin D1 and VEGF, despite PUGNAc treatment. *p < 0.05.

 
 
Figure 7 – O-GlcNAcylation of β-catenin decreases its transcriptional activity while S23 mutation negates this change. DU145 cells were co-transfected with TOPflash and FOPflash reporter constructs. pRenilla Luciferase served as the transfection control. Data set was normalized to pRenilla. Transcriptional activity of WT and pEGFP tagged β-catenin decreased upon PUGNAc treatment while there was no change in DU145 cells transfected with  S23G β-catenin. Preliminary Data. *p<0.10.

 
Figure 8 – Wnt activation increases β-catenin levels and influences  O-GlcNAcylation of β-catenin in a time and dose dependent manner DU145 cells treated with 150 and 300 ng/ml Wnt3A for 4, 8, 12 hour time points. A) Levels of β-catenin increased in a time dependent manner. B) Du145 cells treated with 150 and 300 ng/ml of Wnt3A were precipitated with WGA-agarose.  At 150ng/ml, Wnt3A treament caused a steady increase in O-GlcNAc modified β-catenin (12hour) while treatment of DU145 cells with 300ng/ml caused a drastic decrease (12hour). There seems to be a threshold level of Wnt activation required in the regulation of O-GlcNAc-β-catenin.

 
Figure 9PI3K inhibition increases O-GlcNAcylation of β-catenin in a dose dependent manner. Whole cell lysate of DU145 cells treated with 50 or 100nM of LY294002 were probed with β-catenin antibodies or precipitated with WGA-agarose. Upon LY294002 treatment, levels of O-GlcNAcylated β-catenin increased at 100nM independent of time. PI3K inhibition was ensured as phosphorylated(P)-AKT, a defined marker of an activated PI3K pathway, levels decreased while total AKT levels remain unchanged.

 

Figure 10 – O-GlcNAcylation of endogenous β-catenin affects its localization in U2OS but not SAOS2. A) and B) U2OS and SAOS2 cell lysates were immunoprecipitated for β-catenin with or without PUGNAc treatment. O-GlcNAcylation of β-catenin was detected using WGA-HRP. There was induction of O-GlcNAcylation of β-catenin in U2OS but not SAOS2. *p<0.05. C) Immunofluorescence analysis of the subcellular localization of endogenous β-catenin in U2OS and SAOS2 Osteosarcoma cell line. In U2OS, endogenous β-catenin is widely dispersed throughout the cytoplasm and the nucleus. Upon PUGNAc treatment, β-catenin localized to the plasma membrane, similar to the observation in DU145. Alternatively, localization of endogenous β-catenin in SAOS2 upon PUGNAc treatment did not affect its localization, similar to the response of DU145 transfected with S23 mutant β-catenin. Nucleus-Blue stained with DAPI. Endogenous β-catenin was visualized using Alexafluor 488 secondary antibody to β-catenin primary antibody.