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: eLife

Article Title: Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4

doi: 10.7554/eLife.48767

Figure Lengend Snippet: ( A ) Immunostaining of NR5A1, GATA4, WT1, SOX9 and DMRT1 (red signal) in fibroblasts (HPF) 3 days post infection. DAPI (blue signal) was used to indicate nucleus. Scale bar = 25 μm. NC represents HPF stained with only secondary antibody. ( B ) Experimental design for the reprogramming of human Sertoli-like cells (hiSCs). Human fibroblasts were infected by lentivirus carrying human transcription factors: NR5A1, GATA4, WT1, SOX9 and DMRT1 (5TFs). The cells were cultured in MEF medium for 1 day after infection and followed by G418 selection for 5 days, then changed to DMEM/F12 medium. hiSCs were characterized 10–14 days post viral transduction. ( C ) Schematic protocol for the enrichment of Sertoli-like cells (hiSCs). An AMH:EGFP reporter was integrated to the fibroblasts for hiSCs quantification and isolation. ( D ) FACS analysis of AMH:EGFP+ cell at day 10 after 5TFs infection. The percentage of EGFP+ cell was used to determine the reprogramming efficiency. Two types of human fibroblasts (HPF and dH1) were tested. Cells infected by p2k7 empty virus were used as negative control (CTRL). ( D ) Morphology of re-plated AMH:EGFP+ hiSCs after FACS. Scale bar = 20 μm. ( F ) The mRNA level of AMH was enriched in AMH:EGFP+ hiSCs, as compared to dH1 infected by p2k7 empty virus (dH1-2K7) and AMH:EGFP-. n = 3, technical replicates of ~5 × 10 4 cells were collected by FACS. All data are presented as means ± SD. *p<0.05, **p<0.01, Student’s t-test.>3 independent experiments were carried out. ( G ) Immunofluorescent staining of KRT18 in AMH:EGFP+ hiSCs and dH1 infected by p2k7 empty virus (dH1-2K7) after FACS. Scale bar = 100 μm.

Article Snippet: Antibody , anti-human WT1 (Rabbit polyclonal) , Proteintech , Cat#: 12609–1-AP , IF(1:100).

Techniques: Immunostaining, Infection, Staining, Cell Culture, Selection, Transduction, Isolation, Virus, Negative Control

Journal: eLife

Article Title: Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4

doi: 10.7554/eLife.48767

Figure Lengend Snippet: ( A ) Quantitative PCR result showing the mRNA level of GATA4 , DMRT1 , NR5A1 , SOX9 and WT1 was overexpressed in 293FT cells after virus infection. ACTIN was used as the housekeeping gene for normalization. n = 2 (technical replicates of ~4 × 10 5 cells were collected). All data are presented as means ± SD, *p<0.05, **p<0.01, Student’s t-test.>2 independent experiments were carried out. ( B ) hiSCs showed epithelial morphology change after 5 TFs overexpression. Magnified images were shown on the right side. Two types of cell density were tested. Scale bars = 400 μm. ( C ) Quantitative PCR result showing that the mRNA level of epithelial markers increased after overexpression of NR5A1, GATA4, SOX9, WT1 and DMRT1 (5TFs). Fold expression was normalized to the group infected by empty virus p2k7 (CTRL). ACTIN was used as the housekeeping gene for normalization. All data are presented as means ± SD. *p<0.05, **p<0.01, Student’s t-test. n = 2 (technical replicates of ~4 × 10 5 cells were tested). ( D ) Quantitative PCR result showing the mRNA levels of Sertoli cell markers. Relative expression of 5TFs groups was normalized to the level of HPF cells infected by empty virus 2K7 (CTRL). All data are presented as means ± SD, asterisks indicate statistical significance, (*p<0.05, **p<0.01, Student’s t-test).

Article Snippet: Antibody , anti-human WT1 (Rabbit polyclonal) , Proteintech , Cat#: 12609–1-AP , IF(1:100).

Techniques: Real-time Polymerase Chain Reaction, Virus, Infection, Over Expression, Expressing

Journal: eLife

Article Title: Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4

doi: 10.7554/eLife.48767

Figure Lengend Snippet: ( A ) Representative FACS results of different combinations of NR5A1, GATA4, WT1, SOX9 and DMRT1 for the induction of AMH:EGFP+ cells. dH1 fibroblasts were transduced with the indicated factors and reprogrammed for 10 days. The combinations were divided into three groups: -NR5A1, combinations without NR5A1; +NR5A1, combinations with NR5A1 but without GATA4; +NR5A1 and GATA4, combinations with both NR5A1 and GATA4. ( B ) Quantitative data of EGFP+ cells in ( A ). n = 2, biological replicates, error bar indicates SD, three independent combination experiments were conducted. ~10 4 cells were analyzed in each experiment. ( C ) Immunofluorescent staining of KRT18 in 2F-hiSCs and dH1-2K7 after FACS. Scale bar = 100 μm. ( D ) Heat map of RNA-seq data illustrating differentially co-expressed genes. Red indicates upregulated expression, whereas green indicates downregulated expression. The genes were divided into three groups: Genes upregulated in 2F-hiSCs (dH1), 5F-hiSCs (dH1) and aSCs; genes upregulated in 5F-hiSCs (dH1) and aSCs but downregulated in 2F-hiSCs (dH1); genes downregulated in 2F-hiSCs (dH1), 5F-hiSCs (dH1) and aSCs; all compared to dH1-2K7. The gene number of each group was listed next to the map. Functional enrichment terms of each group and the representative genes were shown on the right side.

Article Snippet: Antibody , anti-human WT1 (Rabbit polyclonal) , Proteintech , Cat#: 12609–1-AP , IF(1:100).

Techniques: Transduction, Staining, RNA Sequencing, Expressing, Functional Assay

Journal: eLife

Article Title: Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4

doi: 10.7554/eLife.48767

Figure Lengend Snippet:

Article Snippet: Antibody , anti-human WT1 (Rabbit polyclonal) , Proteintech , Cat#: 12609–1-AP , IF(1:100).

Techniques: Transfection, Construct, Recombinant, Expressing, Isolation, Software, Staining

Journal: Experimental Animals

Article Title: Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease

doi: 10.1538/expanim.21-0054

Figure Lengend Snippet: The relationship between accumulation of ΔmtDNA in the kidney and podocyte number. (a) Overall scatter plot. In control mice (n=3), the median number of WT1 positive cells per glomerulus was 6.2 cells. The number of WT1 positive cells was decreasing in mito-miceΔ with the proportion of ΔmtDNA exceeding a median of 81.7%. (b) Comparing medians of two groups, high proportion of ΔmtDNA group and low proportion group. The WT1-positive podocyte number was statistically significant decreasing in the group with higher proportion of ΔmtDNA in kidney (median 6.1 cells vs median 4.5 cells, P =0.029). B6 control (n=3), mito-miceΔ (n=8).

Article Snippet: Primary antibodies were anti-aquaporin 1 (AQP1) antibody (polyclonal rabbit IgG, 1:400 dilution; Chemicon International, Temecula, CA), anti-Tamm-Horsfall glycoprotein (THP) antibody (goat antiserum, 1:1,000 dilution; MP Biomedicals, Solon, OH, USA), and anti- Wilms tumor 1 (WT1) antibody (polyclonal rabbit IgG, 1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control

Journal: Experimental and Therapeutic Medicine

Article Title: Resveratrol alleviates obesity-associated podocyte injury in ovariectomized obese rats

doi: 10.3892/etm.2019.8178

Figure Lengend Snippet: Immunohistochemical staining for (A) nephrin and (B) WT-1 in glomeruli (magnification, ×400; scale bar: 100 µm). Semiquantitative analysis for (C) nephrin and (D) WT-1. Data were presented as the mean ± standard deviation (n=7–9 per group). S+N, sham-operation with standard diet, S+H, sham-operation with high-fat diet, O+H, ovariectomy with high-fat diet, O+H+R, ovariectomy plus high-fat diet treated with resveratrol; WT-1, Wilms' tumor-1. *P<0.05 vs. S+N group, ∆ P<0.05 vs. O+H group.

Article Snippet: The primary antibodies used in the present study were: Rabbit anti-tumor necrosis factor α (TNF-α; cat. no. ab9755), rabbit anti-interleukin 6 (IL-6; cat. no. ab25107), rabbit anti-monocyte chemotactic protein-1 (MCP-1; cat. no. ab25124), rabbit anti-nephrin (cat. no. ab136894) and rabbit anti-Wilms' tumor-1 (WT-1; cat. no. ab180840) (all from Abcam, Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Standard Deviation, Wilms Tumor Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Fibrosis of the Neonatal Mouse Heart After Cryoinjury Is Accompanied by Wnt Signaling Activation and Epicardial‐to‐Mesenchymal Transition

doi: 10.1161/JAHA.115.002457

Figure Lengend Snippet: The neonatal heart atrial cardiomyocytes and epicardial cells are Wnt responsive. Neonatal heart of Axin2‐lacZ mice at P1. A, Whole‐mount X‐gal staining of the neonatal heart. Scale bar=0.5 mm. B, X‐gal staining of the epicardial and subepicardial space. B’, Inlet enlarged from (B). C, X‐gal staining of atrial cells. D, P1 neonatal heart of Axin2‐lacZ mice showing Wnt‐responsive cells of valves. B through D, Scale bar=10 μm. E through K, Immunofluorescent staining to characterize Axin2‐positive cells using Axin2‐Cre ERT 2; mTmG neonatal heart labeled at P0 and traced for 2 days. E, Immunostaining against vimentin (red) and GFP (green) in the epicardium. Arrowheads indicate double‐positive cells. F, Immunostaining against Wilms tumor 1 (Wt1) and GFP . Arrowhead indicates a double‐positive cell. G, Immunostaining against cTnT and GFP in subepicardial cardiomyocytes. The arrow indicates a GFP ‐positive epicardial cell not positive for cTnT . The arrowhead indicates a double‐positive cell. H, Immunostaining against cTnT and GFP in atria. I, Immunostaining against vimentin and GFP in the cardiac interstitium. Arrowhead indicates double‐positive interstitial cells. J, Immunostaining against vimentin and GFP in the endocardium. Arrowhead indicates double‐positive cells. E through J, Scale bar=20 μm. K, Immunostaining against vimentin and GFP in a lower magnification stitching image. Scale bar=1 mm. cTnT indicates cardiac troponin T; GFP, green fluorescent protein; P1, postnatal day 1.

Article Snippet: The following antibodies were used: chicken anti–green fluorescent protein (anti‐GFP; 1:1000; Abcam), rabbit antivimentin (1:800; Abcam), mouse anti–cardiac troponin C (1:50; ThermoScientific), rabbit anti–fibroblast‐specific protein 1 (1:100; Abcam), mouse anti–Wilms tumor 1(1:50; Dako), mouse anti–α‐smooth muscle actin (anti–α‐SMA; 1:800; Sigma‐Aldrich) and rat anti‐Ki67 (1:400; eBiosciences).

Techniques: Staining, Labeling, Immunostaining, Wilms Tumor Assay