Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 1. 4integrin, 1integrin, and VCAM-1 in HEK293 cells with stable expression of the Wt1(KTS) protein relative to “empty” pCB6 vector- transfected cells. The mRNAs were quantified by reverse transcription real time PCR and normalized to GAPDH transcripts (A). Asterisks indicate signifi- cant differences between Wt1- and pCB6-transfected cells (Student’s t test with p 0.001, n 7). Increased expression of 4integrin in the Wt1-trans- fected cells was confirmed by immunoblotting with specific antibodies (B).

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Expressing, Plasmid Preparation, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 2. A, effect of Wt1(KTS) proteins on the transcriptional activity of 4integrin promoter reporter con- structs. The different luciferase reporter vectors were transiently co-transfected into HEK293 cells along with expression plasmids for the Wt1(KTS) and Wt1(KTS) proteins. The luciferase activities were normalized to -galactosidase in each sample. Values are means S.E. of five transfection experiments each performed in duplicate.AsterisksindicatesignificantdifferencesversustherespectivepCB6vectorcontrols(Student’sttest, p0.001).TheWt1(KTS)-responsiveelement(s)couldbemappedtotwodistinctregionsbetween283and 171 bp and between 104 and 1 bp relative to the transcription start site in the 4integrin promoter. B, DNA sequence of the 5-flanking region of the human 4integrin gene (NCBI accession number L26059). The two Wt1 binding motifs (Wt1(oligoA) and Wt1(oligoB)) are indicated in bold. The major transcriptional start site is designated “1” (according to Ref. 46). luc, luciferase.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Activity Assay, Luciferase, Transfection, Expressing, Sequencing, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 3. A, electrophoretic mobility shift assay demonstrating binding of Wt1(KTS) proteins to two predicted oligonucleotides (oligoA and oligoB) in the proximal 4integrin promoter. Introducing single base pair mutations into each one of the two oligonucleotides abrogated physical interaction with the Wt1 proteins. Binding of the Wt1(KTS) molecule to the 4integrin promoter sequence could be competed by incubation with the previously identified Wt1 consensus motif from the vitamin D receptor promoter (42), which was used at 1-, 10-, and 100-fold molar concentrations. B, sequences of oligonucleotides that were used for the gel shift experiments.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Incubation, Gel Shift

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 4. A, luciferase reporter assay demonstrating that the two identified Wt1 binding sites can mediate activation of the 4integrin promoter by the Wt1(KTS) protein. The indicated 1.9-kb reporter constructs were tran- siently co-transfected into HEK293 cells along with Wt1(KTS) and Wt1(KTS) expression plasmids. Shown are luciferase activities that were normalized to -galactosidase in each sample. Note that introducing single base pair mutations in oligoA and oligoB (see Fig. 3B) abrogated stimulation of the 4integrin promoter by Wt1(KTS). Values are means S.E. of five experiments each performed in duplicate. Asterisks indicate significant differ- encesversustherespectivepCB6vectorcontrols(Student’sttest,p0.001). B, chromatin immunoprecipitation assay demonstrating binding of Wt1 pro- tein to the 4integrin promoter in K562 leukemia cells. PCR-amplified prod- ucts of the immunoprecipitates were electrophoresed in a 1.5% agarose gel and stained with ethidium bromide; for better visualization the gel photo- graph is presented as a negative of the original. Apparently Wt1 binds to the 5-promoter but not to the 3-region of the 4integrin gene. Input DNA (1:10 dilution) and immunoprecipitates obtained with acetylated histone 3 anti- body (Acetyl H3) served as positive controls. Additional negative control experiments were performed with normal rabbit serum instead of specific antibodies. luc, luciferase.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Luciferase, Reporter Assay, Binding Assay, Activation Assay, Construct, Transfection, Expressing, Chromatin Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 5. Adhesion of HEK293 cells with forced expression of the Wt1(KTS) protein and with stable integration of the pCB6 plasmid. Suspensions of single HEK293 cells were seeded at a density of 5 104 cells/well either in untreated 96-well plates or in dishes that had been coated with the 4integrin ligand VCAM-1. Thirty minutes after seeding, the adherent cells were identified by their characteristic flat shapes (arrows in the micrographs). More than 500 cells in 12 optical fields were counted under the microscope. Note that stable transfection of Wt1(KTS) enhanced cell adhesion to VCAM-1-coated plates, and this effect was reversed upon preincubation with an inhibitory anti-4integrin antibody (VCAM-1 4integrin Ab). Symbols indicate significant differences (p 0.001, analysis of variance with Bonferroni test as posthoc calculation) in cell adhesion between the Wt1(KTS)- and pCB6-transfected cells (*) and between cells that had been treated or not with an inhibitory anti-4integrin antibody (#). Values are means S.E. of three experiments each performed in duplicate.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Expressing, Plasmid Preparation, Microscopy, Stable Transfection, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 6. Double immunofluorescent labeling of 4integrin (green, Cy2 conjugate) and Wt1 (red, Cy3 conjugate) in the heart of a mouse embryo at 11.5 days postcoitus. The images are representative for the more than 20 tissuesectionsthatwereanalyzedfromfourdifferentanimals.Co-localization of both proteins is evident in the developing epicardium but not in myocar- dial cells, which contain neither Wt1 nor 4integrin at a detectable level. The nuclei were counterstained in blue with 4,6-diamidino-2-phenylindole (DAPI) in all panels except one. neg., negative.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Labeling

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 7. Expression analysis of 4integrin, 1integrin, endo B cytokera- tin (K18), and VCAM-1 in the hearts and livers of wild-type (Wt1/) and Wt1-deficient murine embryos (Wt1/) at E12.5. The transcripts were measured by real time RT-PCR and normalized to GAPDH mRNA in each sam- ple. The number of embryos that were analyzed is indicated at the bottom of each bar. Asterisks indicate significant differences in Wt1/ versus normal (Wt1/) embryos. A p value of less than 0.05 was considered statistically significant (Student’s t test).

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrinGene

doi: 10.1074/jbc.m602668200

Figure Lengend Snippet: FIGURE 8. Representative immunoperoxidase staining of 4integrin and vimentin in the heart of a wild-type (Wt1/) and a Wt1-deficient (Wt1/) murine embryo at E11.5. 4integrin can be readily seen in epicar- dialcellsofthenormalembryo(aandc)butishardlydetectableintheWt1/ mutant (e and g). Note that the mesenchymal protein vimentin, which is expressed in the epicardium of the normal embryo (b and d), was also present in the remaining epicardial cells of the Wt1/ mutant (f and arrows in h). Panels g and h represent higher magnifications of the boxed areas in panels e and f, respectively. No staining was obtained with the use of normal goat serum instead of specific antibodies (not shown). Scale bars indicate 50 m.

Article Snippet: For this purpose, the slides were incubated with goat polyclonal anti- 4integrin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; C-20, catalog number sc-6589, Santa Cruz Biotechnology) or with goat polyclonal anti-vimentin antibody (1:50 dilutions in PBS, 0.1% Triton X-100, 3% BSA; catalog number sc-7557, Santa Cruz Biotechnology).

Techniques: Immunoperoxidase Staining, Mutagenesis, Staining