Journal: Molecular Cancer Research

Article Title: Menin Promotes the Wnt Signaling Pathway in Pancreatic Endocrine Cells

doi: 10.1158/1541-7786.mcr-07-2206

Figure Lengend Snippet: FIGURE 1. Expression and colocalization of menin, h-catenin, and TCF3/4 in endocrine and nonendocrine cell lines. A. Western blot shows protein expression of endogenous h-catenin, activated (dephospho)h-catenin, and TCF3/4 in various cell lines transfected with either empty vector or pCMV- SPORT-menin (designated and +, respectively). Forty-eight hours after transient transfection with either control vector or a menin-expressing plasmid (pCMV-Sport-menin), plated cells (HEK 293T, TGP-61, and InR1G9) were harvested and the protein lysate was immunoblotted and probed with antibodies to h-catenin, activated h-catenin (recognize h-catenin dephosphorylated on Ser37 or Thr41), and TCF3/4. Anti-menin antibody was used to confirm overexpression of menin in the pCMV-Sport-menin –transfected cells, and anti –h-tubulin was used to show equal protein loading. For each protein detected such as menin, all images from different cell lines were from the same Western blot and exposure, but the images were separated for publication purposes. B. TGP-61 mouse pancreas islet tumor cells expressing fluorescent epitope–tagged expression constructs show colocalization of menin with h-catenin and TCF3/4. ECFP images are pseudocolored red to distinguish these images from Hoechst dye nuclear staining. ECFP-h-catenin is predominantly nuclear due to overwhelming the proteasome degradation pathway by high levels of ECFP-h-catenin protein expression, but some cytoplasmic protein is still visualized. All images were obtained with 63 objective.

Article Snippet: Mouse islet tumor cells (TGP-61 from American Type Culture Collection) were cultivated in 50% DMEM/50% F12K medium (Mediatech) supplemented with 10% fetal bovine serum and L-glutamine/ penicillin/streptomycin.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Over Expression, Construct, Staining

Journal: Molecular Cancer Research

Article Title: Menin Promotes the Wnt Signaling Pathway in Pancreatic Endocrine Cells

doi: 10.1158/1541-7786.mcr-07-2206

Figure Lengend Snippet: FIGURE 2. Menin promotes TCF luciferase reporter gene activity by activation of the Wnt/h-catenin pathway in TGP-61 mouse islet cells. All experiments were done with triplicate (n = 3) transfections, with the results graphed as the mean F SD. In all of the experiments, the amount of plasmid DNA transfected was kept constant by adding empty vector DNA as needed for each sample. All samples were transfected with the control vector pRL-hTK (Renilla luciferase) to adjust the results for transfection efficiency. A. Overexpression of menin promotes TCF luciferase gene reporter activity when cotransfected with h-catenin. The TCF luciferase reporter vector M50 (Super8XTOPflash) was cotransfected with pCMV-SPORT-menin alone, pCMV-SPORT-h-catenin alone, pCMV-SPORT-h-catenin/pCMV-SPORT-menin, or activated h-catenin mutant (S37A)/pCMV-SPORT-menin. The control sample was transfected with the reporter vector M50 with added empty pCMV-SPORT plasmid DNA. TCF reporter gene assay activity was measured by the Dual Luciferase Assay (Promega) and the results of the firefly luciferase/Renilla luciferase (FL/RL) ratio are graphed for each sample set. Expression of transfected constructs for each experiment is shown on the right. Protein loading was confirmed by h-tubulin expression. B. The augmentation of TCF luciferase gene reporter activity by menin is abrogated by mutation of the TCF enhancer elements of the gene reporter. To prove that activation of the TCF luciferase gene reporter was specifically due to the TCF enhancer, TGP-61 cells were transfected with the TCF reporter vector Super8XTOPflash (M50; label ‘‘T’’ in B) or mutant reporter vector Super8XFOPflash (M51; label ‘‘F’’ in B) either alone or cotransfected with h-catenin– or h-catenin/menin –expressing plasmids as above. Expression of transfected constructs for each experiment is shown on the right. Protein loading was confirmed by h-tubulin expression. C. Menin expression knockdown abrogates the TCF luciferase gene reporter response to overexpressed activated h-catenin. To show that menin knockdown inhibited h-catenin activation of the TCF gene reporter (M50), TGP-61 cells were transfected with either siRNA (Dharmacon) specifically optimized for mouse menin ‘‘M’’ or control ‘‘C’’ siRNA 24 h prior the transfection with the reporter vector M50 and either empty vector or activated h-catenin mutant (S37A)–expressing plasmid. Control sample has reporter vector and siRNA. Expression of transfected constructs for each experiment is shown on the right. Protein loading was confirmed by h-tubulin expression.

Article Snippet: Mouse islet tumor cells (TGP-61 from American Type Culture Collection) were cultivated in 50% DMEM/50% F12K medium (Mediatech) supplemented with 10% fetal bovine serum and L-glutamine/ penicillin/streptomycin.

Techniques: Luciferase, Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Control, Over Expression, Mutagenesis, Reporter Gene Assay, Expressing, Construct, Knockdown