Journal: PLoS Genetics

Article Title: Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of TCF21 Expression at the 6q23.2 Coronary Heart Disease Locus

doi: 10.1371/journal.pgen.1003652

Figure Lengend Snippet: In silico allele-specific transcription factor binding to rs12190287.

Article Snippet: Protein concentrations were determined using a BCA assay (Pierce) and 50–100 μg nuclear protein for each condition was loaded on a pre-cast NuPAGE 4–12% Bis-Tris polyacrylamide gel (Invitrogen/Life Technologies), with gel run at 150 v for 1 h using MES buffer (Invitrogen/Life Technologies), and transferred to PVDF membrane at 35 v for 1 h. Membranes were blocked in 5% non-fat dry milk in 1× TBST for 1 h and incubated overnight with rabbit polyclonal antibodies against TCF21 (Sigma; 0.25 μg/ml), cJUN (Santa Cruz; 1.0 μg/ml), JUND (Santa Cruz; 1.0 μg/ml), ATF3 (Santa Cruz; 1.0 μg/ml), or WT1 (Santa Cruz; 1.0 μg/ml), followed by incubation in a secondary anti-rabbit HRP-conjugated antibody (Invitrogen/Life Technologies; 0.2 μg/ml) and detection by standard ECL (Pierce).

Techniques: In Silico, Binding Assay

Journal: PLoS Genetics

Article Title: Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of TCF21 Expression at the 6q23.2 Coronary Heart Disease Locus

doi: 10.1371/journal.pgen.1003652

Figure Lengend Snippet: ( a ) Dual-luciferase assay of rs12190287 C/G enhancer transfected with human WT1-B (−KTS), WT1-D (+KTS) expression constructs in A7r5 cells, measured after 24 hours. *P<0.01 versus C-Luc or G-Luc+Empty. ( b ) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with human c-JUN and WT1-B or WT1-D expression constructs in A7r5 cells. AP1-Luc reporter was used as a positive control. *P<0.05 versus C-Luc or G-Luc+cJun. ( c ) Dual-luciferase assay of rs12190287 C/G enhancer transfected in heterozygous HCASMC with siRNA against WT1 (−/+KTS) compared to negative control (Neg si). *P<0.05 versus C-Luc or G-luc+Neg si. ( d ) TaqMan based qRT-PCR results showing relative human WT1 mRNA expression levels in heterozygous HCASMC treated with TGF-β1 or PDGF-BB for the indicated times. ( e ) Total enrichment of WT1 at rs12190287 enhancer, FOSB or MYOG promoter regions determined by chromatin immunoprecipitation (ChIP) in heterozygous HCASMC treated with PDGF-BB for 6 hrs. Values represent fold change relative to enrichment with IgG control. *P<0.01 versus Control WT1. ( f ) Allele-specific enrichment of WT1 at rs12190287 determined by HaploChIP in heterozygous HCASMC treated with PDGF-BB, shown as normalized allelic-ratio C/G. *P<0.0005 versus Control WT1. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.

Article Snippet: Protein concentrations were determined using a BCA assay (Pierce) and 50–100 μg nuclear protein for each condition was loaded on a pre-cast NuPAGE 4–12% Bis-Tris polyacrylamide gel (Invitrogen/Life Technologies), with gel run at 150 v for 1 h using MES buffer (Invitrogen/Life Technologies), and transferred to PVDF membrane at 35 v for 1 h. Membranes were blocked in 5% non-fat dry milk in 1× TBST for 1 h and incubated overnight with rabbit polyclonal antibodies against TCF21 (Sigma; 0.25 μg/ml), cJUN (Santa Cruz; 1.0 μg/ml), JUND (Santa Cruz; 1.0 μg/ml), ATF3 (Santa Cruz; 1.0 μg/ml), or WT1 (Santa Cruz; 1.0 μg/ml), followed by incubation in a secondary anti-rabbit HRP-conjugated antibody (Invitrogen/Life Technologies; 0.2 μg/ml) and detection by standard ECL (Pierce).

Techniques: Luciferase, Transfection, Expressing, Construct, Positive Control, Negative Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of TCF21 Expression at the 6q23.2 Coronary Heart Disease Locus

doi: 10.1371/journal.pgen.1003652

Figure Lengend Snippet: Individuals carrying risk alleles for rs12190287 or rs12524865 at 6q23.2 are expected to have increased TCF21 expression upon stimulation of PDGFR-β by PDGF-BB in coronary artery smooth muscle cells, due to increased enrichment of active histone modifications (represented by closed and open diamonds) leading to an open chromatin conformation, allowing binding of an active AP-1 TF complex containing various combinations of c-Jun, JunD, and ATF3. WT1 functions as a transrepressor of this active complex at rs12190287, whereby WT1 may fine-tune the spatial and temporal activation of TCF21 expression. Multiple kinases other than mitogen-activated kinase (MEKK) may be involved in the activation and recruitment of AP-1 complexes to these binding sites.

Article Snippet: Protein concentrations were determined using a BCA assay (Pierce) and 50–100 μg nuclear protein for each condition was loaded on a pre-cast NuPAGE 4–12% Bis-Tris polyacrylamide gel (Invitrogen/Life Technologies), with gel run at 150 v for 1 h using MES buffer (Invitrogen/Life Technologies), and transferred to PVDF membrane at 35 v for 1 h. Membranes were blocked in 5% non-fat dry milk in 1× TBST for 1 h and incubated overnight with rabbit polyclonal antibodies against TCF21 (Sigma; 0.25 μg/ml), cJUN (Santa Cruz; 1.0 μg/ml), JUND (Santa Cruz; 1.0 μg/ml), ATF3 (Santa Cruz; 1.0 μg/ml), or WT1 (Santa Cruz; 1.0 μg/ml), followed by incubation in a secondary anti-rabbit HRP-conjugated antibody (Invitrogen/Life Technologies; 0.2 μg/ml) and detection by standard ECL (Pierce).

Techniques: Expressing, Binding Assay, Activation Assay

Journal: International journal of molecular sciences

Article Title: Porcine Kidney Organoids Derived from Naïve-like Embryonic Stem Cells.

doi: 10.3390/ijms25010682

Figure Lengend Snippet: Figure 5. Gene expression in 3D porcine kidney organoids. (A) Expression of renal progenitor cell markers in the organoids on day 21 and fetal porcine kidney on day 45. Renal progenitor cells markers (EYA1, SIX1) were measured by immunofluorescence test (EYA1 was localized in the cytoplasm, SIX1 was localized in the nucleus). (B) Expression of mature renal cell markers in the organoids on day 21 and fetal porcine kidney on day 45. Mature nephron components markers (PAX2, E-CAD, PODO) were measured by immunofluorescence test and immunohistochemistry test (the circular dotted lines are tubule-like structures; PAX2 was localized in the nucleus; and PODO and ECAD were localized to the cell membrane). (C) Whole-mount co-staining of the organoids for WT1 and CD31 on day 21 (WT1 was localized in the nucleus and CD31 was localized to the cell membrane) (scale bar: 50 µm).

Article Snippet: The primary antibodies included the following: EYA1 (Abcam), SIX1 (Proteintech), PAX2 (Santa Cruz Biotechnology), WT1 (Abcam), E-CAD (Abcam), PODO (Absin), CD31 (Santa Cruz Biotechnology), AQP1 (Proteintech), GLUT1 (proteintech), and KI67 (Abcam); the secondary antibodies included goat anti-rabbit IgG Alexa Fluor 546 (ABclonal), goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-mouse IgG Alexa Fluor 546 (Thermo Fisher Scientific), goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-rabbit IgG H&L(HRP) (Abcam), and goat anti-mouse IgG(H + L) (KeyGEN BioTECH, Nanjing, China).

Techniques: Gene Expression, Expressing, Immunohistochemistry, Membrane, Staining

Journal: Cell stem cell

Article Title: A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery

doi: 10.1016/j.stem.2022.06.005

Figure Lengend Snippet: Key Resources Table

Article Snippet: The following probes from ThermoFisher were used for transcriptional analyses: WT1 (Hs01103751_m1) , MAFB (Hs00534343_s1) , PAX2 (Hs01057416_m1) , HNF4A (Hs00230853_m1) , GATA3 (Hs00231122_m1) , SLC3A1 (Hs00942976_m1) , SLC12A1 (Hs00165731_m1) and SLC12A3 (Hs01027568_m1). . e) Western Blot To prepare protein lysate samples, organoids and control samples were suspended and homogenized in lysis buffer (RIPA buffer (Pierce, 89901) supplemented with 1 mM benzamidine hydrochloride (TCI America, TCB0013), 1X protease inhibitor cocktail (Cell signaling, 5871), 100 μM PMSF (Sigma-Aldrich, 11359061001), and protease inhibitor cocktail tablets (one tablet/10ml of buffer) (Sigma, 11836170001)), and left on ice for 30mins.

Techniques: Plasmid Preparation, Recombinant, TUNEL Assay, Mutagenesis, Software

Journal: Cell stem cell

Article Title: A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery

doi: 10.1016/j.stem.2022.06.005

Figure Lengend Snippet: Key Resources Table

Article Snippet: The following probes from ThermoFisher were used for transcriptional analyses: WT1 (Hs01103751_m1) , MAFB (Hs00534343_s1) , PAX2 (Hs01057416_m1) , HNF4A (Hs00230853_m1) , GATA3 (Hs00231122_m1) , SLC3A1 (Hs00942976_m1) , SLC12A1 (Hs00165731_m1) and SLC12A3 (Hs01027568_m1). . e) Western Blot To prepare protein lysate samples, organoids and control samples were suspended and homogenized in lysis buffer (RIPA buffer (Pierce, 89901) supplemented with 1 mM benzamidine hydrochloride (TCI America, TCB0013), 1X protease inhibitor cocktail (Cell signaling, 5871), 100 μM PMSF (Sigma-Aldrich, 11359061001), and protease inhibitor cocktail tablets (one tablet/10ml of buffer) (Sigma, 11836170001)), and left on ice for 30mins.

Techniques: Plasmid Preparation, Recombinant, TUNEL Assay, Mutagenesis, Software

Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques:

Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: P2Y2R deficiency protects against podocyte loss and glomerular injury in DN. (A) Relative mRNA levels of Nphs1 (nephrin) and Nphs2 (podocin) were determined by real-time PCR analysis (n = 3–5). (B) Localisation of WT1, a podocyte marker, was analysed by immunohistochemistry, and representative images are shown. The number of stained podocytes per glomeruli was counted by using ImageJ software (n = 3). (C) Kidney sections were stained with PAS staining and glomerular morphological changes were scored as described in the method section (n = 3). Data are presented as mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni's multiple comparisons test. ∗∗∗p < 0.001 vs WT control mice; and ### p < 0.001 vs WT DN mice. Scale bar, 50 μm.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques: Real-time Polymerase Chain Reaction, Marker, Immunohistochemistry, Staining, Software, Control

Journal: EMBO Molecular Medicine

Article Title: Inhibition of O‐GlcNAcylation protects from Shiga toxin‐mediated cell injury and lethality in host

doi: 10.15252/emmm.202114678

Figure Lengend Snippet: A Representative immunofluorescence images of the nephron structures within kidney organoids derived from hiPSCs on day 18 of the differentiation; proximal tubules (LTL, green), parietal epithelial cells (PAX8, purple), kidney glomerular podocytes (NPHS1, red), and nephron progenitors (WT1, purple and SIX2, green). Cell nuclei were stained with DAPI. Scale bars: 50 µm. B Representative images showing morphological collapses in iPSC‐derived human kidney organoids following Stx2a (10 ng/ml) treatment for 72 h in the presence or absence OSMI‐1 (10 µM, final). C, D ELISAs were used to analyze the inhibitory effect of OSMI‐1 (10 µM, final) on kidney injury molecule‐1 (KIM‐1) secretion (C) and IL‐8 and CCL‐2 production (D) in culture supernatants of iPSC‐derived human kidney organoids exposed to Stx2a (10 ng/ml) for 72 h ( n = 3 biological replicates). The effects of OSMI‐1 were compared with those of the vehicle (DMSO) controls. E, F Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC‐derived human kidney organoids following Stx2a (10 ng/ml) treatment for 72 h in the presence or absence of OSMI‐1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure ( n = 2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data. Data information: Error bars for bar graphs are presented as mean ± SEM. Statistical analysis was performed using two‐tailed Student’s t ‐test. * P < 0.05; ** P < 0.01; and *** P < 0.001. Source data are available online for this figure.

Article Snippet: WT1 , 1:200 , Cell Signaling Technology 83535 , Alexa Fluor ® 647‐conjugated Donkey Anti‐Rabbit IgG , Jackson ImmunoResearch 711‐605‐152i.

Techniques: Immunofluorescence, Derivative Assay, Staining, Ab Array, Control, Fluorescence, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Inhibition of O‐GlcNAcylation protects from Shiga toxin‐mediated cell injury and lethality in host

doi: 10.15252/emmm.202114678

Figure Lengend Snippet: Primary and secondary antibodies for immunostaining kidney organoids (Subramanian et al , 2019 ).

Article Snippet: WT1 , 1:200 , Cell Signaling Technology 83535 , Alexa Fluor ® 647‐conjugated Donkey Anti‐Rabbit IgG , Jackson ImmunoResearch 711‐605‐152i.

Techniques: Immunostaining, Plasmid Preparation

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: