Journal: Frontiers in Oncology

Article Title: circ0093740 Promotes Tumor Growth and Metastasis by Sponging miR-136/145 and Upregulating DNMT3A in Wilms Tumor

doi: 10.3389/fonc.2021.647352

Figure Lengend Snippet: circ0093740 is upregulated in Wilms tumor compared to normal kidney tissue. (A) The heat map depicted the differentially expressed circRNAs of three paired Wilms tumor and normal kidney tissue. (B) The relative expression level of top five upregulated circRNA between Wilms tumor tissues and normal kidney tissues. (C) The relative expression level of circ0093740 in normal HEK293T cell line and Wilms tumor cell lines. (D) The circular structure of circ0093740 was tested RNase R assay in SKNEP1 Wilms tumor cell line. (E) Circular transcripts of circ0093740 were more stable than its linear PDCH15 mRNA transcripts determined by actinomycin D treated assay in SKNEP1 Wilms tumor cell line. **p < 0.01.

Article Snippet: SKNEP1 Wilms tumor cells were exposed to 5 ug/ml actinomycin D (MCE) to degrade the linear mRNA transcription at 0-, 8-, 16-, and 24-hour time point.

Techniques: Wilms Tumor Assay, Expressing

Journal: Frontiers in Oncology

Article Title: circ0093740 Promotes Tumor Growth and Metastasis by Sponging miR-136/145 and Upregulating DNMT3A in Wilms Tumor

doi: 10.3389/fonc.2021.647352

Figure Lengend Snippet: Suppression of circ0093740 inhibits the proliferation of Wilms tumor cells. (A) siRNA knockdown of circ0093740 was validated in SKNEP1 and G401 Wilms tumor cell line. (B) CCK-8 assays were conducted to evaluate cell proliferation in SKNEP1 Wilms tumor cell line. (C) Mouse xenograft models of SKNEP1 Wilms tumor cell line was established. Tumor volume was estimated in every four days. (D) The images of Ki-67 IHC expression are presented. **p < 0.01.

Article Snippet: SKNEP1 Wilms tumor cells were exposed to 5 ug/ml actinomycin D (MCE) to degrade the linear mRNA transcription at 0-, 8-, 16-, and 24-hour time point.

Techniques: Wilms Tumor Assay, Knockdown, CCK-8 Assay, Expressing

Journal: Frontiers in Oncology

Article Title: circ0093740 Promotes Tumor Growth and Metastasis by Sponging miR-136/145 and Upregulating DNMT3A in Wilms Tumor

doi: 10.3389/fonc.2021.647352

Figure Lengend Snippet: Suppression of circ0093740 inhibits the metastasis of Wilms tumor cells. (A) Wound healing assays were conducted in SKNEP1 and G401 Wilms tumor cell line. (B) Transwell assay was performed to investigate the migration ability of SKNEP1 and G401 Wilms tumor cell line. (C) The number of lung metastases was counted and recorded. HE-stained tumor sections of lung metastases were presented. **p < 0.01.

Article Snippet: SKNEP1 Wilms tumor cells were exposed to 5 ug/ml actinomycin D (MCE) to degrade the linear mRNA transcription at 0-, 8-, 16-, and 24-hour time point.

Techniques: Wilms Tumor Assay, Transwell Assay, Migration, Staining

Journal: Frontiers in Oncology

Article Title: circ0093740 Promotes Tumor Growth and Metastasis by Sponging miR-136/145 and Upregulating DNMT3A in Wilms Tumor

doi: 10.3389/fonc.2021.647352

Figure Lengend Snippet: circ0093740 serves as a sponge of miR-136/145 in Wilms tumor. (A) 18S, ACTIN, circ0093740 and PCDH15 expression in nuclear and cytoplasmic fractions were analyzed by RT-qPCR. (B) miRNA sequencing on three pairs of circ0093740 overexpression and vector samples was conducted in SKNEP1 cell line. (C) Predicted binding sites of miR-136/145 within the circ0093740 sequence. (D) miR-136 and miR-145 expression in Wilms tumor cell lines compared to HEK293T. (E) Luciferase reporter assay was conducted in SKNEP1 and G401 Wilms tumor cell. miR-136 and miR-145 mimics were cotransfected with circ0093740 wild type/mutant luciferase reporter. (F) MS2-based RIP assay transfected with MS2bs-circ0093740 vector, MS2bs-circ0093740-mt vector or Rluc control vector. **p < 0.01.

Article Snippet: SKNEP1 Wilms tumor cells were exposed to 5 ug/ml actinomycin D (MCE) to degrade the linear mRNA transcription at 0-, 8-, 16-, and 24-hour time point.

Techniques: Wilms Tumor Assay, Expressing, Quantitative RT-PCR, Sequencing, Over Expression, Plasmid Preparation, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Transfection, Control

Journal: Frontiers in Oncology

Article Title: circ0093740 Promotes Tumor Growth and Metastasis by Sponging miR-136/145 and Upregulating DNMT3A in Wilms Tumor

doi: 10.3389/fonc.2021.647352

Figure Lengend Snippet: circ0093740 facilitates Wilms tumor progression through circ0093740-miR-136/145-DNMT3A axis. (A) According to TargetScan online website, DNMT3A was the putative downstream target of both miR-136 and miR-145. (B) DNMT3A expression in Wilms tumor cell lines. (C) Expression of DNMT3A transcript was decreased after transfection with miR-136 and miR-145 mimics, detected by qPCR analysis. (D) Luciferase reporter assay of SKNEP1 and G401 Wilms tumor cells co-transfected with miR-136/145 mimics and the 3’-UTR of DNMT3A wild type/mutant luciferase reporter. (E) Enrichment of circ0093740, DNMT3A and miR-136/145 on AGO2 assessed by RIP assay. (F) Enrichment of DNMT3A to AGO2 was increased after silence of circ0093740. (G) Knockdown of circ0093740 contributed to the reduction of DNMT3A protein expression in SKNEP1 and G401 Wilms tumor cell. (H) The molecular mechanism of Circ0093740 Facilitates Wilms Tumor Progression Through circ0093740-miR-136/145-DNMT3A Axis diagram. **p < 0.01.

Article Snippet: SKNEP1 Wilms tumor cells were exposed to 5 ug/ml actinomycin D (MCE) to degrade the linear mRNA transcription at 0-, 8-, 16-, and 24-hour time point.

Techniques: Wilms Tumor Assay, Expressing, Transfection, Luciferase, Reporter Assay, Mutagenesis, Knockdown

Journal: Frontiers in Oncology

Article Title: Primary extraovarian adult granulosa cell tumor of the greater omentum: a case report and literature review

doi: 10.3389/fonc.2025.1689815

Figure Lengend Snippet: Pathological characteristics of the extraovarian adult granulosa cell tumor of the greater omentum. (A–D) Photomicrograph of the tumor (H&E stain). (A) The tumor is encapsulated (H&E stain, 40×magnification, Scale bar: 625 μm). (B) Neoplastic cells are arranged in cord-like and ribbon-like patterns (H&E stain, 100×magnification, Scale bar: 200 μm). (C) Call-Exner bodies are visible (H&E stain, 200×magnification, Scale bar: 100 μm). (D) The tumor is composed of round, oval, or polygonal cells with ill-defined cytoplasmic borders imparting a syncytial appearance. The scant cytoplasm ranges from pale to eosinophilic. Nuclei are round, oval, or angular with finely dispersed chromatin and inconspicuous nucleoli. Longitudinal nuclear grooves and coffee-bean shaped nuclei are present(H&E stain, 400×magnification, Scale bar: 50 μm). (E–I) Photomicrograph of immunohistochemical staining. Immunohistochemistry staining is positive for α-inhibin (E) , SF-1 (F) , FOXL2 (G) , WT-1 (H) . (I) Ki-67 Proliferation Index was 20% (IHC stain, 200× magnification, Scale bar: 100 μm).

Article Snippet: WT-1 , Positive , Strong,diffuse nucleus , Supports diagnosis , OTIRIH(Rabbit Monoclonal), Zhongshan Golden Bridge.

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry

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: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: hrMSCs are enriched in the stroma underlying and adjacent to fallopian tube STIC lesions. A, Representative H&E-stained tissue sections of normal fallopian tubes and fallopian tubes harboring STIC lesions. Histology is paired with multispectral IF images of each respective group shown. Black and white images denote cells with the phenotypes of interest. Red x’s indicate hrMSCs (WT1 + /CD73 + /CD90 + /CD105 + /CD45 − ), whereas yellow x’s indicate nMSCs (WT1−/CD73 + /CD90 + /CD105 + /CD45 − ). B, Schematic showing the criteria for the Vectra spatial quantification. C, hrMSC abundance normalized to the area of ROI. D, hrMSC to nMSC ratios. E, hrMSC abundance (normalized to the area of ROI) in the expanded normal FT patient cohorts consisting of WT ( n = 10 young, n = 9 aged), BRCA1 mutant ( n = 9), or BRCA2 mutant ( n = 6) fallopian tubes lacking STIC/HGSOC. F, Ratio of hrMSC/nMSC in normal FTs. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons analysis. Data points for C – F are reflective of individual ROIs. Statistics were determined on field ROIs for C and D . Statistics were determined with patient averages for E and F . G, Heatmap with unsupervised clustering of STIC stroma vs. normal stroma. H, Enrichment score of DEGs between STIC stroma vs. normal stroma (left: all, right: top 30 genes) applied to stromal location [from normal to STIC distal (dist) to STIC adjacent that contains both STIC contiguous and adjacent regions (STIC adj) to directly underlying STIC to invasive stroma]. I, Volcano plot of significantly differentially expressed genes in STIC stroma vs. normal stroma. J, Enrichment score of WT1 targets in stroma applied to stromal locations as in I . K, Correlation of stromal WT1 enrichment score with epithelial WT1 enrichment score.

Article Snippet: Lentiviral particles encoding PRKAA1 -m-GFP (RC218572L2V), empty vector m-GFP control constructs (PS100071V/PS100093V), WT1-A-m-GFP (RC220079L4V), WT1 shRNA m-GFP (TL300442V), and scramble shRNA (TR30021V) were purchased from OriGene.

Techniques: Staining, Mutagenesis

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: hrMSCs exhibit the epigenetic and phenotypic profile of CA-MSCs. A, DNA methylation array represented as a heatmap. MSCs were taken from benign patient tissues (nMSCs; black circles; n = 20), BRCA1/2 mut carrier patients without cancer (red circles; n = 10), and patients with confirmed invasive HGSOC (CA-MSC; purple circles; n = 14). B, UMAP of MSCs derived from patients in A . C, Relative fluorescence intensity (RFI) of WT1 405 was determined by flow cytometry for WT ( n = 5), BRCA1 mut ( n = 3), and BRCA2 mut ( n = 13) MSC cell lines. WT1 405 RFI was plotted against patient age. Linear regression of WT1 405 MFI vs. patient age (gray line); R 2 = 0.3745. D, MSCs were categorized into age groups. E, MSCs were classified into nMSCs or hrMSCs based on WT1 405 RFI with a minimum cutoff of 2 RFI for hrMSCs. This categorization was independent of BRCA status and age. F and G, Representative histograms and violin plots of nMSC and hrMSC WT1 expression. H and I, Ovarian tumor cells (TC) were grown independently or in coculture with nMSCs, hrMSCs, or CA-MSCs under adherent ( H ) or nonadherent ( I ) conditions. Individual cells and spheroids per low powered field (LPF) were counted and graphed ( n = 3). J, Tumor cell adherence to nMSCs, hrMSCs, and CA-MSCs is shown ( n = 3). For D–J , P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparison analysis. K and L, WT1 MFI determined by flow cytometry following coculture of nMSCs or hrMSCs in monoculture or coculture with FTE or ovarian cancer cells. M, WT1 KD and scramble shRNA control hrMSCs were generated by lentiviral transduction and validated by flow cytometry. N–P, Relative fold change (FC) of tumor cells after coculture with WT1 shRNA KD or scramble shRNA hrMSCs under ( N ) adherent or ( O ) nonadherent conditions. P, Chemoresistance assay on the aforementioned cocultures. Q – T, Similarly, WT1 OE or empty vector (EV) nMSCs were generated using lentiviral transduction. Tumor cells were cocultured with transduced lines, and tumor cell proliferation, sphere formation, and chemoresistance were assessed.

Article Snippet: Lentiviral particles encoding PRKAA1 -m-GFP (RC218572L2V), empty vector m-GFP control constructs (PS100071V/PS100093V), WT1-A-m-GFP (RC220079L4V), WT1 shRNA m-GFP (TL300442V), and scramble shRNA (TR30021V) were purchased from OriGene.

Techniques: DNA Methylation Assay, Derivative Assay, Fluorescence, Flow Cytometry, Expressing, Comparison, shRNA, Control, Generated, Transduction, Plasmid Preparation

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: Transformed, metastasized P53 null FTE harbor mutational hallmarks of HGSOC. Whole-genome sequencing was used to characterize mutations present in transformed P53 null FTE originating from P53 null FTE/hrMSC organoids. A, Oncoprint including mutations in genes commonly mutated in HGSOC. This includes correlations to COSMIC single- and double-base signatures that are shown [single-base substitution (SBS) and double-base substitution (DBS), respectively]. B, Summary statistics of mutational analysis including somatic synonymous and nonsynonymous mutations. C, Representative graph of single-base mutations for one sample exhibiting the pan-cancer mutational signature (signature 5). D, Gene set enrichment analysis on STIC stroma relative to normal stroma from DSP. E, Enrichment score of stromal genes associated with oxidative stress–induced senescence derived from our DSP dataset. F, Gene set enrichments shared between STIC stroma and STIC epithelium. G, IF and ( H ) flow cytometry of hrMSCs stained with the general oxidation probe CellROX Green and CellROX DR, respectively. Representative cells are shown at 20× magnification with a 100 μm scale bar. In G and K , individual data points correspond to individual nuclei. More than 3 fields per group were taken to analyze 150–200 individual cells. In H – J , individual data points correspond to separate wells. I, Flow cytometry analysis of MSC CellROX DR after treatment with the antioxidant Trolox. J, Flow cytometry analysis of carboxyfluorescein succinimidyl ester (CSFEL)-labeled FTE cells cocultured with MSCs ( n = 3). MFI data were normalized for cell number. FTE CellROX values are displayed. K, 53BP1 foci per FTE nuclei after 24-hour coculture with hrMSCs ±10 μmol/L Trolox. L, Simple linear regression correlating WT1 relative fluorescence intensity (RFI) with CellROX DR determined by flow cytometry. Individual data points represent single cells. M, CellROX DR MFI following lentiviral overexpression of WT1 in nMSCs. N, CellROX DR MFI following lentiviral shRNA KD of WT1 in hrMSCs. O, FTE or ovarian cancer cells were cocultured with either CellTrace-labeled ( O ) nMSCs or ( P ) hrMSCs. MSCs were assessed for changes in CellROX DR fluorescence. For G – P , P values were determined by the Student‘s t test. Q, WT1 overexpression induces MSC and FTE oxidative stress, resulting in increased FTE DNA DSBs.

Article Snippet: Lentiviral particles encoding PRKAA1 -m-GFP (RC218572L2V), empty vector m-GFP control constructs (PS100071V/PS100093V), WT1-A-m-GFP (RC220079L4V), WT1 shRNA m-GFP (TL300442V), and scramble shRNA (TR30021V) were purchased from OriGene.

Techniques: Transformation Assay, Sequencing, Derivative Assay, Flow Cytometry, Staining, Labeling, Fluorescence, Over Expression, shRNA

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: Increased oxidative stress in hrMSCs results in increased lipid peroxide breakdown products capable of inducing DNA DSBs in FTE. 4-HNE and MDA were compared between nMSCs and hrMSCs by fluorescence microscopy ( n = 3) and flow cytometry ( n > 3; A–J ). Representative images at 20× magnification are shown. More than three fields were imaged and analyzed per condition. Silicon rhodamine (SiR) actin stain was used for cell masking. In B and G , each data point represents an individual cell, whereas in D and I , each data point represents the MFI of individual patient cell lines ( n = 3 nMSC lines and n = 7 hrMSC lines). C and H, Stacked histograms of nMSCs, hrMSCs, and hrMSCs with 10 μmol/L Trolox treatment as a negative control. E and J, Flow cytometry was utilized to conduct a simple linear regression correlating WT1 expression with either MDA or 4-HNE. Individual points represent single cells. K, Representative images of MDA in hrMSCs (CTV + ) that are cocultured with FTE (CT CSFE + ). White arrowheads depict MDA puncta that are visible within nanotubule connections between cells ( n > 3). L and M, Analysis of BODIPY C-12/CellTrace DR double-positive FTE after coculture with BODIPY C12-stained hrMSCs. hrMSCs were treated with 5 μmol/L Lat B or ethanol control overnight preceding coculture. Individual data points represent individual wells. Black circles represent FTE alone, and green circles are FTE with hrMSC coculture. %Double positive indicates FTE that received hrMSC-derived lipids. Quantification of ( N ) MDA-APC and ( O ) 4-HNE-PE MFIs in FTE cell monocultures or cocultured with nMSCs or hrMSCs. P , FTE were treated with 5–10 μmol/L 4-HNE and assayed for 53BP1 foci. Student t test was used to determine significance in all panels except P , where P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparison analysis. Q, Excessive stromal lipid peroxidation contributes to increased DNA damage in FTE.

Article Snippet: Lentiviral particles encoding PRKAA1 -m-GFP (RC218572L2V), empty vector m-GFP control constructs (PS100071V/PS100093V), WT1-A-m-GFP (RC220079L4V), WT1 shRNA m-GFP (TL300442V), and scramble shRNA (TR30021V) were purchased from OriGene.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Staining, Negative Control, Expressing, Control, Derivative Assay, Comparison

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: Aging-associated AMPK derepression of the JNK/c-JUN/WT1 axis results in the hrMSC protumorigenic phenotype. A, DNA methylation in nMSCs, hrMSCs, and CA-MSCs at the promoter region of the PRKAG1 gene. B, qRT-PCR expression data of AMPK-related genes in nMSC vs. hrMSC. C, Hypothesized AMPK/JNK/c-JUN/WT1 axis. D – F, Characterization of phospho-AMPKα1 and total AMPKα1, JNK, and c-JUN in nMSCs and hrMSCs. Representative Western blots are shown with corresponding densitometries. Patient samples were grouped, and statistical significance was determined using the Student’s t test. For both nMSCs and hrMSCs, N > 3 patients. G and H, Simple linear regressions correlating p-JNK and p-c-JUN with WT1 expression on a per-sample basis via densitometry. I, Quantification of AMPK Western blot levels in nMSC vs. hrMSC at passage 4 vs. passage 8. J, Western blot of hrMSCs treated with increasing doses of the JNK inhibitor SP600125. Quantified band intensities are shown. K and L, Western blot and quantification of AMPKα1 and pAMPKα1 following BC1618 treatment for 24 hours. M, BC1618-treated hrMSCs were analyzed by flow cytometry for MDA fluorescence. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparison analysis. M – P, Quantification of p-JNK, p-c-JUN, and WT1 bands following treatment of hrMSCs with either BC1618 or SP600125. Q, Representative 20× images of FTE that were cocultured at a 1:1 ratio with hrMSC AMPKα1-m-GFP or empty vector (EV) transduced. The percentage of cells with >9 53BP1 foci was quantified by fluorescence microscopy. More than 3 fields per condition were taken to analyze 150–200 individual cells. The Student’s t test was used to determine the significance of Q . R, Aging-associated loss of AMPK phosphorylation and expression results in the derepression of JNK phosphorylation, resulting in increased WT1 expression, oxidative stress, and FTE DNA DSBs.

Article Snippet: Lentiviral particles encoding PRKAA1 -m-GFP (RC218572L2V), empty vector m-GFP control constructs (PS100071V/PS100093V), WT1-A-m-GFP (RC220079L4V), WT1 shRNA m-GFP (TL300442V), and scramble shRNA (TR30021V) were purchased from OriGene.

Techniques: DNA Methylation Assay, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Comparison, Plasmid Preparation, Microscopy, Phospho-proteomics

Journal: Journal of the American Society of Nephrology

Article Title: A Novel Missense Mutation of Wilms’ Tumor 1 Causes Autosomal Dominant FSGS

doi: 10.1681/asn.2013101053

Figure Lengend Snippet: Figure 7. Overexpression of WT1R458Q impairs focal contact assembly in podocytes. (A) Expression of vasodilator-stimulated phosphoprotein (VASP) is confirmed by immunoblot analysis in immortalized podocytes under growth-permissive and growth- restrictive conditions. (B) Representative images of immortalized human podocytes overexpressing WT1WT or WT1R458Q im- munostained for the focal contact protein VASP. Note the im- paired formation focal contacts in WT1DR458Q-overexpressing cells (inset) relative to those seen in tGFP-overexpressing controls or WT1DWT-overexpressing cells.

Article Snippet: Plasmids expressing tGFP-tagged human WT1WT were purchased from OriGene (Rockville, MD).

Techniques: Over Expression, Expressing, Western Blot