Journal: Cancers

Article Title: HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1

doi: 10.3390/cancers11101436

Figure Lengend Snippet: Biological consequences of HDAC3 inhibition and interplay between β-catenin, MYC, and WT1. ( A ) MV4-11 cells were pretreated with z-VAD-FMK for 1 h, followed by incubation with RGFP966 as indicated for 24 h. Cells were fixed and stained with PI and analyzed by flow cytometry ( n = 3; mean + SD; two-way ANOVA; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). ( B ) MV4-11 cells were treated as indicated for 24 h. After incubation time, cells were stained with annexin-V-FITC/PI and apoptosis was determined by flow cytometry ( n = 3; mean + SD; two-way ANOVA; ** p ≤ 0.01). ( C ) MV4-11 cells were treated with increasing doses of RGFP966 (1 µM, 5 µM, and 10 µM) for 24 h. Caspase-3, PARP1, and WT1 (→ marks WT1 cleavage product) were analyzed by Western blot, with vinculin as loading control ( n = 2). ( D ) MV4-11 cells were treated as stated in ( C ). Indicated proteins were detected via Western blot, with GAPDH as loading control ( n = 2). ( E ) Immunofluorescence for 53BP1 in MV4-11 cells that remained untreated or exposed to 1 µM RGFP966 for 24 h. ( F ) MV4-11 cells were treated as indicated for 24 h. Thereafter, cells were harvested and washed with PBS and reseeded. After 4 days, the cell numbers were determined by trypan blue staining and counting ( n = 3; mean + SD; one-way ANOVA; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: We used siRNAs at the following concentrations: β-catenin (100 pmol; Cell Signaling Technology, Frankfurt, Germany, #6225), MYC (100 pmol; Santa Cruz, Heidelberg, Germany, sc-29226), and WT1 (100 pmol; Santa Cruz, Heidelberg, Germany, sc-36846).

Techniques: Inhibition, Incubation, Staining, Flow Cytometry, Western Blot, Control, Immunofluorescence

Journal: Cancers

Article Title: HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1

doi: 10.3390/cancers11101436

Figure Lengend Snippet: Cross-talk between β-catenin, MYC, and WT1. ( A ) MV4-11 cells were electroporated with 100 pmol of indicated siRNAs and incubated for 48 h (NC siRNA, scrambled irrelevant control siRNA; si, siRNA). Proteins were detected by Western blot, with HSP90 as loading control ( n = 3). ( B ) Densitometry evaluation of data shown in (A) ( n = 3; mean + SD; two-way ANOVA; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001). ( C ) Flow cytometry was done with aliquots of MV4-11 cells with an efficient elimination of β-catenin, MYC, or WT1. Cells were fixed, PI-stained, and assessed for apoptotic DNA fragmentation ( n = 3; two-way ANOVA; not significant).

Article Snippet: We used siRNAs at the following concentrations: β-catenin (100 pmol; Cell Signaling Technology, Frankfurt, Germany, #6225), MYC (100 pmol; Santa Cruz, Heidelberg, Germany, sc-29226), and WT1 (100 pmol; Santa Cruz, Heidelberg, Germany, sc-36846).

Techniques: Incubation, Control, Western Blot, Flow Cytometry, Staining

Journal: Nature communications

Article Title: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases.

doi: 10.1038/s41467-019-10729-5

Figure Lengend Snippet: Fig. 3 Similar subcellular localization of WT1 with YAP. a Immunofluorescence staining for WT1 in epithelial cell sheets on flat substrata and NRA. Translocation of WT1 into nuclei was observed in marginal zones and FLPs of sheets expanding on NRA (brown boxes). Submarginal cells on NRA (red boxes) and on flat substrata showed YAP in the cytoplasm. The samples were fixed after 8 h to remove stencils. b Control and YAPKD cells and their nuclear fractions were analyzed using immunoblotting using YAP and WT1 antibodies. c Immunofluorescence staining for WT1 in cells and d immunoblotting of YAP and WT1 abundance in the nuclei in different density–epithelial cell sheets showing density-dependent translocation of WT1 into the nucleus

Article Snippet: For each transfection, 6 μl of WT1 siRNA (Santa Cruz, sc-36846), TAZ siRNA (Thermo Fisher, 122501), and unconjugated control siRNA-A (Santa Cruz, sc-37007) in 100 μl of transfection medium (Santa Cruz, sc-36868) were mixed with 6 μl of transfection reagent (sc-29528) in another 100 μl of transfection medium.

Techniques: Staining, Translocation Assay, Control, Western Blot

Journal: Nature communications

Article Title: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases.

doi: 10.1038/s41467-019-10729-5

Figure Lengend Snippet: Fig. 4 YAP regulates E-cadherin through WT1 in epithelial layers on NRA. a Immunofluorescence staining for WT1 in YAPKD cell sheets (top), and for YAP in WT1KD cell sheets cultured on NRA. The samples were fixed after 8 h to remove stencils. b Co-IP analysis using the YAP antibody, followed by immunoblotting using the WT1 antibody. c Chromatin immunoprecipitation (ChIP) analysis of the WT1-YAP complex binding at the E-cadherin promoter (n = 3). We extracted cross-linked chromatin and immunoprecipitated it using antibodies against YAP, WT1, IgG (negative control), and RNAPII or histone H3 (see Supplementary Fig. 12a) antibodies (positive controls). The immunoprecipitated chromatin and input genomic DNA were used for amplification of the E-cadherin promoter, CTGF promoter (known to be regulated by YAP but not WT1, and used as another control), and GAPDH promoter (n = 4 biologically independent samples, * = statistical significance of PCR products from each sample vs. IgG, *P < 0.05 and **P < 0.01). d E-cadherin expressions of control and WT1KD cells analyzed by immunoblotting with WT1 and E-cadherin antibodies (n = 3 biologically independent samples, * = statistical significance of E-cadherin expression of control and WT1KD cells, ***P < 5 × 10−3). e Cell migration speed of individual cells in control and WT1KD epithelial cells in the marginal region of the sheets in the presence of an E-cadherin blocking antibody (each number of independently analyzed cells, n, is indicated, * = statistical significance, n.s = no significance, and ***P < 5 × 10−3). f Dissemination of cells in control and WT1KD epithelial sheets on NRA in the presence of an E-cadherin blocking antibody (n = 4 biologically independent experiments, * = statistical significance, *P < 0.05, ***P < 5 × 10−3, and n.s = no significance). g Immunoblotting of phosphorylated YAP and total YAP in cell sheets cultured in the presence of different concentrations of E-cadherin blocking antibody (n = 3 biologically independent samples). h Schematic of the YAP-mediated cell dissemination triggered by mechanical cues stemming from NRA, operating through E-cadherin control by YAP-WT1 complexes and leading to cell dissemination in epithelial cell sheets on NRA. All error bars are S.E.M and statistical significance was determined by two-sided Student’s t-test

Article Snippet: For each transfection, 6 μl of WT1 siRNA (Santa Cruz, sc-36846), TAZ siRNA (Thermo Fisher, 122501), and unconjugated control siRNA-A (Santa Cruz, sc-37007) in 100 μl of transfection medium (Santa Cruz, sc-36868) were mixed with 6 μl of transfection reagent (sc-29528) in another 100 μl of transfection medium.

Techniques: Staining, Cell Culture, Co-Immunoprecipitation Assay, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Control, Expressing, Migration, Blocking Assay

Journal: Molecular Medicine Reports

Article Title: miR-590 regulates WT1 during proliferation of G401 cells

doi: 10.3892/mmr.2017.6561

Figure Lengend Snippet: miR-590 was upregulated in Wilms' tumor tissue. (A) Quantitative polymerase chain reaction analysis of miR-590 expression levels in Wilms' tumor tissue (n=65), the adjacent normal tissue (n=65) and normal kidney (n=20). The relative quantity of miR-590 was normalized to U6. (B) Expression levels of miR-590 in different clinical tumor stages of Wilms' tumor. The relative quantity of miR-590 was normalized to U6. All data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01. miR, micro RNA.

Article Snippet: When cells reached 60% confluence, they were transfected with miRNA mimic, si-RNA WT1 (cat. no. sc-36846; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or expression vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Wilms Tumor Assay, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: miR-590 regulates WT1 during proliferation of G401 cells

doi: 10.3892/mmr.2017.6561

Figure Lengend Snippet: miR-590 targeted WT1 and its expression. (A) Schematic presentation of the binding site of miR-590 in the 3′-UTR of WT1 and WT1 3′-UTR luciferase reporter constructs, including wild-type and mutant type. (B) Validation of the transfection of miR-590 by using reverse transcription-quantitative polymerase chain reaction. (C) Dual-luciferase reporter assays indicated that in the wild-type miR-590 expression was reduced, and in the mutant, the WT1 3′-UTR binding site was affected, thus activity in G401 cells was inhibited following transfection with miR-590 mimic. (D) Western blot analysis of WT1 expression level in cultured G401 cells transfected with miR-590 (10 nM) or miR-NC. Quantitative analysis of WT1 protein level was normalized to β-tubulin. All data are presented as the mean ± standard deviation from three independent experiments. *P<0.01 vs. miR-control. CMV, cytomegalovirus; WT1, Wilms' tumor 1; 3′-UTR, 3′-untranslated region; miR, microRNA.

Article Snippet: When cells reached 60% confluence, they were transfected with miRNA mimic, si-RNA WT1 (cat. no. sc-36846; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or expression vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Expressing, Binding Assay, Luciferase, Construct, Mutagenesis, Biomarker Discovery, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot, Cell Culture, Standard Deviation, Control, Wilms Tumor Assay

Journal: Molecular Medicine Reports

Article Title: miR-590 regulates WT1 during proliferation of G401 cells

doi: 10.3892/mmr.2017.6561

Figure Lengend Snippet: Importance of WT1 in G401 cell proliferation using siRNA. G401 morphology was determined by fluorescence microscopy. Proliferation was assessed using an EdU cell proliferation assay kit. G401 cells were stained by EdU and DAPI 36 h after transfection. Cell proliferation rate was increased following overexpression of si-RNA-WT1. Magnification, ×100. siRNA, small interfering RNA; WT1, Wilms' tumor 1; EdU, 5-ethynyl-20-deoxyuridine.

Article Snippet: When cells reached 60% confluence, they were transfected with miRNA mimic, si-RNA WT1 (cat. no. sc-36846; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or expression vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Fluorescence, Microscopy, Proliferation Assay, Staining, Transfection, Over Expression, Small Interfering RNA, Wilms Tumor Assay

Journal: Molecular Medicine Reports

Article Title: miR-590 regulates WT1 during proliferation of G401 cells

doi: 10.3892/mmr.2017.6561

Figure Lengend Snippet: Western blot analysis of WT1 expression level in cultured G401 cells. Quantitative analysis of WT1 protein levels was normalized to β-tubulin. WT1 was successfully expressed in G401 cells. PCDNA-3.1 and PCDNA-3.1-WT1 plasmids (500 or 1,000 ng) were transfected into G401 cells with Lipofectamine 2000. WT1, Wilms' tumor 1.

Article Snippet: When cells reached 60% confluence, they were transfected with miRNA mimic, si-RNA WT1 (cat. no. sc-36846; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or expression vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Cell Culture, Transfection, Wilms Tumor Assay

Journal: Molecular Medicine Reports

Article Title: miR-590 regulates WT1 during proliferation of G401 cells

doi: 10.3892/mmr.2017.6561

Figure Lengend Snippet: Importance of WT1 in G401 cell proliferation following WT1 overexpression. G401 morphology was determined by fluorescence microscopy. Proliferation was assessed using the EdU cell proliferation assay kit. G401 cells were stained by EdU and DAPI 36 h after transfection. Cell proliferation rate was decreased following WT1 overexpression. siRNA, small interfering RNA; WT1, Wilms' tumor 1; EdU, 5-ethynyl-20-deoxyuridine.

Article Snippet: When cells reached 60% confluence, they were transfected with miRNA mimic, si-RNA WT1 (cat. no. sc-36846; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or expression vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Over Expression, Fluorescence, Microscopy, Proliferation Assay, Staining, Transfection, Small Interfering RNA, Wilms Tumor Assay

Journal: Biochemical Journal

Article Title: WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line

doi: 10.1042/bj20101734

Figure Lengend Snippet: Figure 2 Expression of BASP1 in K562 cells leads to the general down-regulation of WT1 target genes

Article Snippet: Control siRNAs (small interfering RNAs) used were from Ambion (AM4611 and AM4635) and WT1 siRNAs were from Santa Cruz Biotechnology (sc-36846) and Ambion (s14912).

Techniques: Expressing

Journal: Biochemical Journal

Article Title: WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line

doi: 10.1042/bj20101734

Figure Lengend Snippet: Figure 5 WT1 is required for the altered PMA-dependent differentiation programme of B-K562 cells

Article Snippet: Control siRNAs (small interfering RNAs) used were from Ambion (AM4611 and AM4635) and WT1 siRNAs were from Santa Cruz Biotechnology (sc-36846) and Ambion (s14912).

Techniques: