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:

Journal: Oncotarget

Article Title: STIM1 plays an important role in TGF-β-induced suppression of breast cancer cell proliferation

doi: 10.18632/oncotarget.7619

Figure Lengend Snippet: A. Assessment of STIM1 mRNA levels by qRT-PCR after TGF-β treatment for 24 and 48 h. B. Assessment of STIM1 protein expression level by Western blot analysis. C. The statistical analysis of STIM1 protein expression level referenced to GAPDH. D. Assessment of WT1 mRNA levels by qRT-PCR after TGF-β treatment for 24 h. E. Assessment of STIM1 mRNA levels by qRT-PCR after WT1 siRNA knockdown and TGF-β treatment. *P<0.05 VS. Control and NC group. F. Assessment of STIM1 protein expression level by Western blot analysis after WT1 siRNA knockdown and TGF-β treatment. G. The statistical analysis of STIM1 protein expression level referenced to GAPDH in different groups. *P<0.05 VS. Control and NC group. Data were representative of three independent experiments and bar graphs represented as the means ± SE. Significance was assessed using one-way ANOVA with Bonferroni's multiple comparisons post-tests, *p < 0.05.

Article Snippet: The WT1 siRNAs and scrambled siRNA were purchased from Shanghai Genepharma RNAi Company (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

doi: 10.1186/1756-9966-30-110

Figure Lengend Snippet: miR-15a/16-1 downregulates WT1 protein level not through targeting mRNAs according to the degree of complementarity with their 3'UTR . (A) K562 and HL-60 cells were transiently transfected with pRS-15/16 or pRS-E vector for different time periods and subjected to western analysis with the indicated antibodies. The level of GAPDH was used as a loading control. (B) K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector for 24 and 48 hours, then the relative expression of WT1 was measured by quantitative real-time PCR. (C and D). K562 and HL-60 cells were transfected with the pGL-3 containing Bcl-2 3'UTR or WT1 3'UTR and pRS-15/16 or pRS-E for 24 hours, relative repression fold of firefly luciferase expression was standardized to Renilla luciferase, pGL-TK.

Article Snippet: SiRNA sequences targeting WT1 (National Center for Biotechnology Information accession number AH003034) were synthesized. siRNA-WT1: ccauaccagugugacuuca corresponds to positions 9-27 of exon 7 within the WT1 coding sequence[ ].

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Real-time Polymerase Chain Reaction, Luciferase

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

doi: 10.1186/1756-9966-30-110

Figure Lengend Snippet: AMO-miR-15a/16-1 reversed the expression of WT1 in K562 and HL-60 cells (A) and (B) AMO inhibited the expression of miR-15a/16-1 . K562 and HL-60 cells were incubated with AMO-miR-15a/16-1 for 24 and 48 hours, then miR-15a/16-1 and U6 snRNA expression were determined by quantitative real-time PCR. (C) and (D) K562 and HL-60 cells were incubated with AMO-miR-15a/16-1 for 48 h, then mRNA and protein level of WT1 were detected by quantitative real-time PCR and Western blotting individually. * P < 0.01 versus SCR.

Article Snippet: SiRNA sequences targeting WT1 (National Center for Biotechnology Information accession number AH003034) were synthesized. siRNA-WT1: ccauaccagugugacuuca corresponds to positions 9-27 of exon 7 within the WT1 coding sequence[ ].

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

doi: 10.1186/1756-9966-30-110

Figure Lengend Snippet: The role of miR-15a/16-1 in the regulation of leukemic cell proliferation . (A) and (B) K562 and HL-60 cells were incubated with 1.5 ug siRNA-WT1, 1.5 ug N.C or neither of the above for 24 and 48 hours, then the relative expression of WT1 and the corresponding WT1 protein level were separately measured by quantitative real-time PCR and Western blotting. (C) and (D) K562 and HL-60 cells treated with siRNA or N.C or neither of the above were measured by CCK-8 assay at different time periods. Data are shown as mean ± SD from three independent experiments. * P < 0.05 versus negative control.

Article Snippet: SiRNA sequences targeting WT1 (National Center for Biotechnology Information accession number AH003034) were synthesized. siRNA-WT1: ccauaccagugugacuuca corresponds to positions 9-27 of exon 7 within the WT1 coding sequence[ ].

Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

doi: 10.1186/1756-9966-30-110

Figure Lengend Snippet: WT1 protein expression is inversely correlated with miR-15a or miR-16-1 expression in AML samples and normal controls . (A) WT1 protein levels from 2 normal controls (N1 and N2) and 6 AML samples (P1-P6) were measured by Western blotting. The numbers represent the relative expression of miR-15a and miR-16-1 in the same specimens. (B) and (C) Inverse correlation between miR-15a or miR-16-1 expression and WT1 protein level in 25 primary AML samples and 5 normal controls. A statistically significant correlation between miR-15a or miR-16-1 expression and WT1 protein level was observed by Pearson's method. WT1 verse miR-15a R = -0.73 P < 0.01; WT1 verse miR-16-1 R = -0.76 P < 0.01

Article Snippet: SiRNA sequences targeting WT1 (National Center for Biotechnology Information accession number AH003034) were synthesized. siRNA-WT1: ccauaccagugugacuuca corresponds to positions 9-27 of exon 7 within the WT1 coding sequence[ ].

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: Cell cycle arrest gene targets of miR-642a-5p. ( a ) Ingenuity Pathway Analysis of cell cycle targets of miR-642a-5p. Green denotes genes downregulated by miR-642a-5p, and the number of seed sites in their 3’UTR (identified by TargetScan 7.2) indicated. Red denotes genes upregulated by miR-642a-5p. ( b ) RT-qPCR analysis of the cell cycle genes following overexpression of miR-642a-5p in 22Rv1 PCa cells. Expression of target mRNAs is normalized to HPRT housekeeping gene expression, calculated using the 2 −ΔΔCt method, and relative to miR-NC. Error bars = SE; n = 3; * p < 0.05 relative to miR-NC. ( c ) Oncomine analysis of the expression of the miR-642a-5p targets in PCa data sets. (i) WT1; (ii) NUAK1; (iii) RASSF3; (iv) SKP2; (v) IGFBP3; and (vi) GPS2. The data cohorts indicated above each graph, and n per group shown. Boxes denote the median (horizontal line); whiskers indicate distances to the highest and lowest values [for NUAK1 and RASSF3 the lower whisker is to the 10th percentile (minimum value removed)]. * p < 0.05, ** p < 0.005.

Article Snippet: Flexitube siRNAs to WT1 were from Qiagen (WT1#1 Cat#SI00008267; WT1#4 Cat#SI00008288; WT1#7 Cat#SI03056298; and WT1#8 Cat#SI03061331).

Techniques: Quantitative RT-PCR, Over Expression, Expressing, Gene Expression, Whisker Assay

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: WT1 is a direct target of miR-642a-5p in PCa cells. ( a ) The 3′UTR of WT1 has three putative miR-642a-5p seed sites as predicted by TargetScan 7.2. ( b ) Schematic of the 3′UTR of WT1 (not to scale). Depiction of the GeneCopoeia target clone, which contains only the first 1293 base pairs of the 3′UTR, is with green shading. The grey shaded boxes indicate the miR-642a-5p seed sites. ( c ) Luciferase reporter gene analysis of the 3′UTR of the putative miR-642a-5p target WT1 in 22Rv1 and LNCaP PCa cells transiently overexpressing miR-642a-5p or miR-NC (20 nM). DOHH and miR-642a-5p perfect targets are positive controls. Error bars = SD; n = 3; ** p < 0.005.

Article Snippet: Flexitube siRNAs to WT1 were from Qiagen (WT1#1 Cat#SI00008267; WT1#4 Cat#SI00008288; WT1#7 Cat#SI03056298; and WT1#8 Cat#SI03061331).

Techniques: Luciferase

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: Targeted siRNA-mediated inhibition of WT1 expression reduces PCa cell proliferation and blocks cell cycle progression. ( a ) Relative cell viability of 22Rv and LNCaP PCa cells measured via cell titer assay at 3 d post-transfection with WT1 siRNA or si-NC (20 nM). Validation of WT1 knockdown see Fig. B. Error bars = SD; n = 3; ** p < 0.005. ( b ) Proliferation of 22Rv1 and LNCaP PCa cells (cell index) measured using the xCELLigence system post WT1 siRNA or si-NC transfection (20 nM). Validation of WT1 knockdown see Fig. C. Error bars = SD; n = 3. ( c ) Colony formation assay of 22Rv1 and LNCaP PCa cells 14–21 days post WT1 siRNA or si-NC (20 nM) transfection. Validation of WT1 knockdown see Fig. C. Error bars = SD; n = 3; ** p < 0.005. ( d ) Flow cytometry cell cycle analysis of 22Rv1 and LNCaP PCa cells transfected with WT1 siRNA or si-NC (20 nM) for 72 h. Validation of WT1 knockdown see Fig. D. n = 3; * p < 0.05, ** p < 0.005 relative to si-NC. ( e ) Western blot analysis of p21 (22Rv1 and LNCaP) and p53 (22Rv1) protein expression 72 h post-transfection of PCa cells with 20 nM WT1 siRNA or si-NC. β-actin is the loading control. Validation of WT1 knockdown see Fig. E. For full-length, non-cropped blots see Fig. B and S1C. n = 3. ( f ) Relative cell viability of 22Rv1 and LNCaP PCa cells measured via cell titer assay at 5 days post-transfection with WT1 siRNA or si-NC (20 nM), and 3 d post-17-AAG treatment (1 µM). Validation of WT1 knockdown see Fig. B. Error bars = SD; n = 3; ** p < 0.005.

Article Snippet: Flexitube siRNAs to WT1 were from Qiagen (WT1#1 Cat#SI00008267; WT1#4 Cat#SI00008288; WT1#7 Cat#SI03056298; and WT1#8 Cat#SI03061331).

Techniques: Inhibition, Expressing, Titer Assay, Transfection, Biomarker Discovery, Knockdown, Colony Assay, Flow Cytometry, Cell Cycle Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: WT1 overexpression increases colony formation and miR-642a-5p rescues this effect. ( a ) RT-qPCR analysis of WT1 gene expression following stable or transient LeGO-iT2-WT1-203 or LeGO-iT2-Empty plasmids, and overexpression of miR-642a-5p or miR-NC in 22Rv1 and LNCaP PCa cells. Expression of WT1 is normalized to HPRT housekeeping gene expression, calculated using the 2 −ΔΔCt method, and relative to Empty vector + miR-NC. Error bars = SE; n = 3; * p < 0.05, ** p < 0.005 relative to Empty vector + miR-NC. # p < 0.05 WT1-203 + miR-NC relative to WT1-203 + miR-642a-5p. ( b ) Colony formation assay of 22Rv1 PCa cells 14 days post transient WT1 overexpression/empty vector transfection and miR-NC/miR-642a-5p (30 nM) co-transfection. Error bars = SD; n = 3; ** p < 0.005 relative to Empty vector + miR-NC. ## p < 0.005 WT1-203 + miR-NC relative to WT1-203 + miR-642a-5p.

Article Snippet: Flexitube siRNAs to WT1 were from Qiagen (WT1#1 Cat#SI00008267; WT1#4 Cat#SI00008288; WT1#7 Cat#SI03056298; and WT1#8 Cat#SI03061331).

Techniques: Over Expression, Quantitative RT-PCR, Gene Expression, Expressing, Plasmid Preparation, Colony Assay, Transfection, Cotransfection