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: 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: Scientific Reports

Article Title: Identification of m6A methyltransferase-related WTAP and ZC3H13 predicts immune infiltrates in glioblastoma

doi: 10.1038/s41598-025-88671-4

Figure Lengend Snippet: Relative expression of genes expression in GBM tissues. Expression levels of WTAP and ZC3H13 genes were compared with those of the control group on GBM tissues, 10 GBM and adjacent tissues, respectively ( A ). The expression levels of siRNA WTAP and overexpression ZC3H13 genes ( B and C ). The expression levels of CD27, CD70, CD80, CD86, ICOS, CTLA4, and LAG3 in siRNA WTAP and overexpression ZC3H13 were demonstrated in Fig. 9D and F, respectively. The result were determined by qRT-PCR. The data are mean ± standard deviation of six replicate experiments. * P < 0.05, **P < 0.01 and ***P < 0.001 compared with the corresponding control values.

Article Snippet: After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: WTAP (A04296-2, Boster Biotech, Wuhan, China), CD27 (A01148-2, Boster Biotech), CD70 (A02853-2, Boster Biotech), CD80 (A00196-3, Boster Biotech), CD86(BM4121, Boster Biotech), ICOS (A00291-2, Boster Biotech), CTLA4 (A00020-1, Boster Biotech), LAG3 (M02869-2, Boster Biotech), Beta-actin (BM3873, Boster Biotech).

Techniques: Expressing, Control, Over Expression, Quantitative RT-PCR, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: APT1-derived depalmitoylation of CD36 alleviates diabetes-induced lipotoxicity in podocytes

doi: 10.7150/ijbs.109220

Figure Lengend Snippet: Construction of db/db model to evaluate changes in renal pathology lipid metabolism. Two groups of twenty-week-old mice were analyzed: db/m (n=6) and db/db (n=6). A-B. Representative PAS staining of glomeruli in each group, and the semiquantitative analysis of mesangial area ratio. (**<0.01, scale bar: 50 μm). C-G. Biochemical examination: (C) UACR, (D) serum creatinine, (E) blood glucose, (F) serum cholesterol, (G) serum triglycerides. (**<0.01). H. Representative transmission electron microscopy (TEM) images of podocyte foot processes and glomerular basement membrane (GBM) in each group. (**<0.01, scale bar: 10 μm). I-J. Representative immunofluorescence double staining of BODIPY and WT1 in the glomeruli of db/m and db/db mice, and semiquantitative analysis. (**<0.01, scale bar:50μm). K-L. Oil Red O staining of lipid droplets (LDs) in the glomeruli of db/m and db/db mice, and semiquantitative analysis. (**<0.01, scale bar: 40 μm).

Article Snippet: Frozen kidney sections were fixed, blocked, and then stained with WT1 antibody (BM4216, Boster, China) for 1 h at 37°C, subsequently incubated in the presence of secondary antibody and BODIPY dye, and finally covered with DAPI.

Techniques: Staining, Transmission Assay, Electron Microscopy, Membrane, Immunofluorescence, Double Staining