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Wnt Snail main point of paper
- Snail interacts with b-catenin to promotes Wnt dependent target gene expression
- Snail acts to Decrease E-cadherin, but is not needed to Increase Wnt activity by binding to B-catenin
- End result of Wnt activation is to increase tumor growth
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Wnt pathway
- Wnt signals attach to: Frizzled protein
- Frizzled protein activates: Disheveled
- Disheveled BLOCKS binding of: GSK-3b
- GSK-3b therefore is not able to phosphorylate: B-catenin
- B-catenin then acts with T-cell factor(TCF) TFs at promoter sequences to aid in transcription
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If Wnt pathway mutated
GSK-3B phosphorylates B-catenin and causes it to degrade
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Fig 1 results
- Main point: Shows that when Snail is TRANSFECTED with B-catenin, LRP6da, Dishevelled2 or endogenous B-catenin in SW480 cells: increases TRANSCRIPTIONAL activation of TCF/B-catenin dependent reporter expression
- TOP(WT binding sites at promoter)/FOP(mutated TCF binding sites) reporter system shows if snail affects the binding to TCF/b-catenin for transcription
- LRP6da and Dishevelled2 ACTIVATE Wnt pathway
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Fig 2 results
- Main point: transcriptional activation by Snail is NOT based on e-cadherin expression or B-catenin stabilization
- Mutants made of Snail with N or C terminal deletions, as well as Zinc finger deletion
- TOP/FOP showed: Zinc finger mutant showed to be important for B-catenin activation of transcription, as it was still repressed with B-catenin present
- Western blot looked at stability of B-catenin: Showed Snail does NOT affect stability of B-catenin, even after activation upstream by LRP6da(activating Wnt). Does NOT affect phosphorylation or nuclear localization of B-catenin either.
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Fig 3 results
- Main point: Snail interacts with B-catenin and N teminal of B-catenin needed for snail binding and increasing promoter activity.
- All snail mutants shown to Co-PRECIPITATE with B-catenin (Co-IP Western blots)
- Snail does NOT co-precip with B-catenin mutant
- Snail Activates GAL4-B-catenin, but NOT GAL4-B-catenin MUTANT (GAL4 used to avoid interference by endogenous B-catenin)
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Fig 4 results
- Main point: Snail is required for Wnt target gene expression
- Doxycycline used to turn ON Snail, and when on, showed INCREASE in Axin2 at protein level and in reporter construct stimulation, looking at Flag-snail in CARCINOMA CELLS
- Axin2: target of Wnt signaling!
- Survivin also used (Wnt signal target) and increased w/SNAIL
- Snail siRNA showed DECREASE in all Wnt target genes at mRNA LEVEL (Survivin, Axin2, etc)
- At protein level, Survivin and Axin reduced w/ Snail siRNA, but B-catenin and E-cadherin levels remain THE SAME w SnailsiRNA. E-cadherin NOT need to be repressed for B-catenin transcription stimulation
- Axin2 promoter activity also REDUCED with Snail siRNA
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