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: Journal of Biomedical Science

Article Title: ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

doi: 10.1186/1423-0127-17-3

Figure Lengend Snippet: Podocytes isolated out of the urine of patients with nephrotic syndrome express ADAM10 . Cells isolated out of the urine of a patient with nephrotic syndrome were analyzed by flow cytometry (A+E) , Western blot (B+C) , RT-PCR (C lower panel) , and immunofluorescence (D+F) . (A) Cells isolated from the urine were stained with ADAM10 or L1 adhesion molecule and analyzed with Cellquest software from Becton Dickinson (Heidelberg, Germany). (B) Urinary cells were lysed and western blots (WB) with ADAM10 and L1 (L1 11A) specific antibodies were performed. (C) Lower panel: RT-PCR with α3, β1, and podocin specific primers on cDNA of cells isolated from the urine. Upper panel: Western blot analysis with α3, β1 and podocin specific antibodies in lysats of cells isolated from the urine. (D) Immunofluorescence double staining of cells isolated from the urine with podocyte specific marker proteins α3, nephrin, podocyin antibodies. Images were documented with a Zeiss camera. (E) Urinary cells were investigated by intracellular FACS staining using WT1 (podocyte specific marker protein) and ADAM10 antibodies. Stained cells were analyzed with Cellquest software from Becton Dickinson (Heidelberg, Germany). (F) Immunofluorescence staining of untreated (control) and IFN-γ treated urinary podocytes with L1 specific primary antibodies followed by Alexa 488 coupled secondary antibodies. Nuclei of urinary podocytes were stained and visualized with DAPI. Images were documented with a Zeiss camera. (G) Glomeruli from human kidney were isolated and glomerular lysats were prepared, proteins were loaded on a SDS gel and western blot analysis were performed using a polyclonal antibody against the cytoplasmic tail of L1.

Article Snippet: WT1 antibody for immunofluorescence analysis was purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Isolation, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Software, Double Staining, Marker, Control, SDS-Gel

Journal: Journal of Biomedical Science

Article Title: ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

doi: 10.1186/1423-0127-17-3

Figure Lengend Snippet: ADAM10 is expressed in podocytes in human renal tissue . (A) Glomeruli from human kidney were isolated and lysed and investigated by an ADAM10 specific westernblot (left panel). Right panel, double immunofluorescence analysis on a human kidney section with WT1 (red) and ADAM10 (green) antibodies, demonstrating ADAM10 expression in WT1 positive podocytes. (B) Increased ADAM10 levels are found in the urine of patients with glomerular kidney diseases . Western Blot analysis of ADAM10 expression in urine and urinary vesicles of healthy volunteers (HV 1-2) and patients with glomerular kidney diseases (number of patients P1-5, ADAM10 expression in supernatants (SN) and vesicles (VES) from untreated (HPC C) or treated with 1 μM ionomycin (HPC IONO) for 24 h. Membranes were reprobed with CD9 an specific marker protein of exosomes.

Article Snippet: WT1 antibody for immunofluorescence analysis was purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Isolation, Immunofluorescence, Expressing, Western Blot, Marker

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: 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: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: The table presents the quantification of tumor cellularity, PD-L1 or CD274 TPS (Tumor Proportion Score), intensity, LI (lymphocyte infiltration) percentage, and contributions of CD4 and CD8 lymphocytes in LI

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Expression of immunosuppressive markers correlates to BRAF mutation. (A) The tumor tissues of 19 PTC cases were stained with CD274 antibody, and the expression intensity was scored by a COH pathologist. Grade 1 represents the lowest expression, and grade 3+ represents the highest. (B) The tumor tissue was processed to detect CD73, and the expression intensity was scored where grade 1 represents the lowest expression and grade 3+ represents the highest. ( C ) The images represent the cells positive for cancer cells (TTF-brown), T cells CD8 (blue) and CD4 (pink) in the two representative tumor tissue of BRAF -mutated and WT samples. ( D ) The plot illustrates the percentage of lymphocyte infiltration within the tumor area, as assessed by the pathologist. The lymphocytic infiltration ratio is notably higher for mutant patients (red dots) compared with wild-type patients (blue). Additionally, the percentages of CD4 and CD8 lymphocytes were analyzed. The findings suggest a higher contribution of CD4 lymphocytes compared with CD8, reinforcing the cell type infiltration data. PTC, papillary thyroid cancer; WT, wild-type. * indicates a p value of <0.05

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Expressing, Mutagenesis, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: BRAF knockdown induced CD274 expression. (A) The RNA extracted from four PTC cell lines was used to determine the expression of BRAF , CD274, CD73, CD200, CD276, ENTPD1, and CD200. The expression in the mutant was compared with the WT cell type. (B) The immunoblot represents the expression of proteins in the BRAF -mutated and WT cell lines. (C) The bar graph represents the changes in the mRNA expression of CD274 and other immunosuppressive markers on knocking down BRAF . (D) Immunoblot represents the knockdown of BRAF and upregulation of CD274 in the BRAF -mutated cell lines T32 and T85 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance; PTC, papillary thyroid cancer; WT, wild-type.

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Knockdown, Expressing, Mutagenesis, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: Cell cycle arrest correlates with CD274 expression. (A, B) The T85 cell line was treated with increasing concentration of BRAF inhibitors, and changes in the proliferation were measured using the live cell imaging system. (C, D) Data representing the proliferation changes in the T68 cell lines with respect to BRAF inhibitor treatment. (E) FACS data representing the changes in the cell cycle stages of T68 with respect to BRAF inhibitors where dabrafenib induced stronger cell cycle arrest. (F) The qPCR data represents the changes in the expression of BRAF , CD274 and CD73 in the T68 and T85 cells with respect to the BRAF inhibitor treatment. ( G ) The immunoblot represents the changes in the CD274 and CD73 expression on combining dabrafenib with either binimetinib, temsirolimus, or abemaciclib (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Expressing, Concentration Assay, Live Cell Imaging, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: BRAF inhibitor-induced transcriptional changes upstream of CD274. (A) Cartoon showing the design of CD274-promoter-luciferase constructs used for determining the changes in transcriptional activity postdrug treatment. (B) The box plot representing the luciferase activity determined in the cells post 48 hours of transfection, where 2 Kb promoter sequence correlated with maximum activity. (C) Luciferase activity measured in the cells post 48 hours of BRAF or KRAS knockdown. ( D ) The bar graph represents the difference in the promoter activity between the T85 and T32 cell lines. (E, F) Luciferase (Promoter) activity correlated with increased BRAF inhibitor concentration in the T68 and T85 cells. (****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Luciferase, Construct, Activity Assay, Transfection, Sequencing, Knockdown, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: The drug combination effect on CD274 expression. (A, B) The bar graph represents the percentage change in the growth of T68 or T85 cells on binimetinib, abemaciclib or temsirolimus exposure. The immunoblot shows the change in the expression of CD274 and CD73 on drug treatment. (C) The bar graph represents the effect of drug combinations on the cell viability of T85 cells as measured by CCK8 assay. (D–F) The immunoblot represents the increase in the expression of CD274 and CD73 expression on combining dabrafenib with either binimetinib or temsirolimus, or abemaciclib (*****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Expressing, Western Blot, CCK-8 Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

doi: 10.1136/jitc-2023-008505

Figure Lengend Snippet: The synergistic effect of BRAF and checkpoint inhibitors in immune cell-mediated killing. (A) The line graph represents the growth kinetics of T85 cells when cultured in combination with activated CD8 cells. The bar graph represents the comparison in growth between monoculture and cocultured T85 cells at 0 and 72 hours time points. (B) The growth kinetics of T32 cells when cultured in combination with activated CD8 cells. (C) The images represent the effect of dabrafenib, CD8, atezolizumab, and IL-2 on the T85 (GFP) cell growth. (D) The growth kinetics of T85 cells when cultured in combination with activated CD8 cells, IL-2, and atezolizumab. (E) The growth kinetics of T85 cells, when cultured in combination with dabrafenib, activated CD8 cells, IL-2 and atezolizumab. (F) The images represent the effect of CD8, atezolizumab, and IL-2 on the T85 (GFP) derived spheroids. Loss of GFP represents a loss of spheroid viability. (G) The bar graph represents the changes in the spheroid viability when cocultured with CD8, IL-2, and atezolizumab. (H) The changes in the spheroid viability when cocultured with dabrafenib, CD8, IL-2, and atezolizumab. (I) The immunoblot represents the changes in the CD274 expression in T85 cells on exposure to IL-2 and dabrafenib n monoculture or coculture. **** indicates P value < 0.001.

Article Snippet: Likewise, the pGL3 plasmid harboring the WT or mutant UTR region of the CD274 gene were purchased from Addgene (#107009, #107010).

Techniques: Cell Culture, Comparison, Derivative Assay, Western Blot, 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 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: