Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: Three-step proteomic approach identifies putative PAX8-interacting partners. (a) Schematic of the workflow for the PAX8-interacting partner’s identification. (b) Representative examples of endogenous PAX8 immunoprecipitation silver-stained gel and immunoblot from fallopian tube secretory cells (FT) and ovarian carcinoma cells (OV). IMR90 cells, which are PAX8 negative, were used as negative control (C). (c) Immunoblot of size-exclusion fractions showing the presence of PAX8 at 600 kDa size range. PAX8 immunoprecipitates and gel-filtration fractions were sent for mass spectrometry analysis. (d) Volcano plot of identified putative PAX8-interacting partners. (e) Confirmation of PAX8 and SOX17 interaction by co-immunoprecipitation experiments from FTSEC and HGSOC cell lines. (f) PAX8 and SOX17 immunoblots of size exclusion fractions demonstrate co-elution at 600 kDa size range fractions.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Immunoprecipitation, Staining, Western Blot, Negative Control, Filtration, Mass Spectrometry, Co-Elution Assay

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 have a strong correlation and dependency on ovarian cancer. (a) TCGA ovarian cancer Pearson correlation of PAX8 with the newly identified PAX8-interacting partners. (b) SOX17 has the strongest correlation with PAX8 in ovarian cancer. (c) Ovarian carcinoma cells PAX8 dependency. (d) Ovarian carcinoma cells SOX17 dependency and (e) Ovarian carcinoma cells PAX8-SOX17 co-dependency.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 physically interacts with SOX17 in the fallopian tubes and in ovarian cancer. (a) Immunohistochemistry showing nuclear co-expression of PAX8 and SOX17 in FTSEC and HGSOC cells. 5 normal samples and 5 ovarian cancer patients were analyzed and one representative case of each is shown. (b) Immunofluorescence showing co-localization of PAX8 and SOX17 in benign cells (FT194, FT246, and FT282) and in malignant cells (OVCAR4, KURAMOCHI, and OVSAHO). (c) Proximity ligation assay signals in secretory cells (FT194, FT246, and FT282) and in carcinoma cells (OVCAR4, KURAMOCHI, and OVSAHO) are shown in red. The nuclei are stained with DAPI (blue). The nuclei were acquired in one z-plane with 60X magnification. (d) In situ proximity ligation assay signals in normal fallopian tube sections and high-grade serous ovarian cancer samples shown in brown and the nuclei in orange. The nuclei were acquired in one z-plane with 40x magnification. *Number of PLA signals (PAX8-SOX17 interactions) was determined by counting 100 nuclei per sample (Supplemental information 2).

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Immunohistochemistry, Expressing, Immunofluorescence, Proximity Ligation Assay, Staining, In Situ

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: Overexpression of PAX8 and SOX17 in ovarian carcinoma cases. (a) Higher expression of PAX8 in ovarian cancer samples. (b) Differential expression of SOX17 in ovarian cancer samples. (c) Most of the TCGA ovarian cancer samples have high PAX8 and SOX17 co-expression. (d) Higher number of PAX8-SOX17 interactions on ovarian cancer cell lines.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Over Expression, Expressing

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 are transcription co-regulators. (a-b) Immunoblot analyses following knockdown of PAX8 or SOX17 in FTSEC (FT194, FT246, and FT282) and in HGSOC (OVCAR4, KURAMOCHI, and OVSAHO) cells showing SOX17 expression dependency on PAX8 regulation. (c-d) Real-time PCR analysis following of PAX8 or SOX17 in FTSEC (FT194, FT246, and FT282) and in HGSOC (OVCAR4, KURAMOCHI, and OVSAHO) cells depicting the transcriptional co-regulation of SOX17 by PAX8. (e-f) Luciferase reporter assay using reporter with 5X PAX8-recognition sequence.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Sequencing

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8-SOX17 expression co-regulation. (a-b) Immunoblot of four deconvoluted siPAX8 and four deconvoluted siSOX17 knockdowns on FTSEC and HGSOC cell lines. (c-d) PAX8 and SOX17 protein levels after knockdowns with four pooled siPAX8, four pooled siSOX17 or then all combined by reverse-phase protein array.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Expressing, Western Blot, Protein Array

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 regulate a common set of genes. (a) RNA-seq unsupervised clustering of PAX8, SOX17, and commonly regulated target genes. Profiles obtained with OVCAR4 after 72 hr knockdowns; fold change >1; P < 0.05. (b) Venn diagram representing number of genes up-regulated under each condition. (c) Ontology analysis of the PAX8-SOX17 commonly up-regulated genes. (d) RPPA unsupervised clustering of PAX8-SOX17 commonly regulated proteins. (e) Top-ranked PAX8-SOX17 commonly regulated proteins. (f) Immunoblot showing SERPINE1 up-regulation after PAX8 or SOX17 knockdown. (g) Real-time PCR showing SERPINE1 up-regulation after PAX8 or SOX17 knockdown.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: RNA Sequencing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8-SOX17 tightly suppresses SERPINE1 expression. (a) RNA-seq analysis depicting SERPINE1 gene up-regulation after PAX8, SOX17 or DUAL knockdown. (b) SERPINE1 average expression after PAX8, SOX17 or DUAL knockdown. (c) RPPA ontology analysis corroborating enrichment of angiogenesis and VEGF pathways. (d) SERPINE1 relative protein levels in different FTSEC lines and HGSOC lines, depicting suppression of SERPINE1 during malignant transformation. (e) Immunoblot of different benign and malignant cell lines showing drastically reduction of SERPINE1 levels in cell lines with increased SOX17 expression.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Expressing, RNA Sequencing Assay, Transformation Assay, Western Blot

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 regulate the secretion of angiogenesis mediators. (a) Human angiogenesis array of conditioned media from FTSEC and HGSOC cells following PAX8 or SOX17 knockdown. (b) Effect of PAX8 knockdown on specific analytes was produced by quantifying the array membrane spots intensity. (c) ELISA for quantification of secreted SERPINE1 in the FTSEC- and HGSOC-conditioned media. (d) ELISA for the quantitation of secreted VEGF in the FTSEC and HGSOC conditioned media. (e) ELISA showing SERPINE1 secreted by FTSEC and HGSOC. (f) ELISA experiments showing the effects of PAX8 or SOX17 knockdown on levels of SERPINE1 in HGSOC.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, Quantitation Assay

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 regulate the secretion of angiogenesis mediators. (a) Human angiogenesis arrays of fresh DMEM/F12 and RMPI media with no detection of angiogenesis mediators, negative controls. (b-c-d) Human angiogenesis array of additional three fallopian tube secretory cells and additional three ovarian carcinoma cells conditioned media after PAX8 or SOX17 knockdown.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 and SOX17 promote ovarian cancer angiogenesis. (a) Endothelial cells tube formation assay. Representative images depict negative control, VEGF, SERPINE1, FTSEC, and HGSOC conditioned media after PAX8 or SOX17 knockdown. (b) Quantitation of the HUVEC neo-vessels loops. (c) Neovascularization in angioreactors containing conditioned media from HGSOC cells, but not from FTSEC after implantation in nude mice. (d) Quantitation of endothelial cell invasion into angioreactors.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Tube Formation Assay, Negative Control, Quantitation Assay

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8-SOX17 regulates angiogenesis through the PLCy1 pathway. (a) Schematic illustration of the PLCy1 pathway. (b) RPPA analysis showing up-regulation of VEGFR2 protein levels followed PAX8 and SOX17 knockdown. (c) RPPA analysis showing reduced phosphorylated-PLCy1 and phosphorylated-ERK1/2 (active molecules) followed PAX8 and SOX17 knockdown. (d) Confirmation by western blot of the PLCy1 pathway inactivation.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Western Blot

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8 or SOX17 doxycycline-inducible knockdown inhibits ovarian cancer progression. (a) Immunoblot of OVTOKO cells harboring inducible shPAX8 or shSOX17 before and after the induction with 1μg/ml of doxycycline. (b) In vivo imaging of OVTOKO tumors in NSG female mice. Animals were imaged after two weeks of doxycycline supplementation. (c) Necropsy of animals depicting ascites and tumors (white arrow) only in the non-targeting control group. (d) The volume of ascites collected from mice after two weeks of doxycycline supplementation. (e) NSG mice reproductive system depicting ovarian cancer volume.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Western Blot, In Vivo Imaging

Journal: bioRxiv

Article Title: PAX8 orchestrates an angiogenic program through interaction with SOX17

doi: 10.1101/2020.09.09.290387

Figure Lengend Snippet: PAX8-SOX17 suppress anti-angiogenesis factors. (a) Spearman correlation analysis of TCGA ovarian cancer data set depicting negative inversely correlation between SOX17 and angiogenesis inhibitors (SERPINE1 and THBS1). (b) Schematic diagram of the activation of tumor angiogenesis by PAX8 and SOX17 expression.

Article Snippet: PLA signals were determined employing Duolink Probes Anti-Mouse MINUS (Sigma-Aldrich: DUO92004) and Anti-Rabbit PLUS (Sigma-Aldrich: DUO92002) as manufacturer’s recommended protocol following overnight incubation with anti-PAX8 mouse antibody 1:250 (Novus: NBP2-29903) and anti-SOX17 rabbit antibody 1:250 (Cell Signaling: 81778S) at 4 °C.

Techniques: Activation Assay, Expressing

Journal: BMC Cancer

Article Title: A genomic and transcriptomic approach for a differential diagnosis between primary and secondary ovarian carcinomas in patients with a previous history of breast cancer

doi: 10.1186/1471-2407-10-222

Figure Lengend Snippet: Comparison between pathological analyses, CGH, transcriptomic profiles, and immunohistochemistry profiles

Article Snippet: Then, the rabbit polyclonal anti-PAX8 antibody (Protein Tech Group Inc., Chicago, IL, USA) was applied (dilution: 1/200), and samples were incubated overnight at 4°C.

Techniques: Comparison, Immunohistochemistry

Journal: Nature Communications

Article Title: PAX8 and MECOM are interaction partners driving ovarian cancer

doi: 10.1038/s41467-021-22708-w

Figure Lengend Snippet: A BioID-MS results from IGROV-1-PAX8-BioID-T2A-mCherry cells. Blue dots represent proteins significantly enriched ( P value <0.01 and Log FC >1). Red dot represents PAX8 and orange dot represents MECOM. B List of transcription factors enriched in PAX8-BioID IP-MS experiment. C Western blot from BioID-WB experiments in two different IGROV-1-PAX8-BioID-T2A-mCherry clones. The picture displays one representative image out of three independent experiments. D Endogenous co-immunoprecipitation between PAX8 and MECOM variants in MFE-319 and IGROV-1 cells. The picture displays one representative image out of three independent experiments. E Co-immunoprecipitation of ectopically expressed PAX8-HA and PRDM3 in HEK293 cells. The picture displays one representative image out of five independent experiments. F NanoBit assay in HEK293A cells transfected with PAX8-LgBit (Lg) and PRDM3-SmBit (Sm). HNF1B and PCBD1 are an unrelated pair used as positive and specificity controls. RLU relative luminescence unit. Data are presented as mean values ± SD from three biological replicates. for Western blots and interaction measurements are provided as a Source Data file.

Article Snippet: Antibodies used are PAX8 (Cell Signaling, 59019, 1:10), PRDM3 (GenScript, U0869CG110-1, 1.5 μg).

Techniques: Protein-Protein interactions, Western Blot, Clone Assay, Immunoprecipitation, Transfection

Journal: Nature Communications

Article Title: PAX8 and MECOM are interaction partners driving ovarian cancer

doi: 10.1038/s41467-021-22708-w

Figure Lengend Snippet: A Schematic representation of PAX8 and corresponding mutants generated by in vitro transcription–translation (IVTT). B IVTT-NanoBit assay testing the interaction of PAX8-LgBit and truncation mutants with PRDM3-SmBit. Data are presented as mean ± SD from four biological replicates. C NanoBit assay testing the interaction of individual PAX8 domains with PRDM3-SmBit. N/C N-terminal and C-terminal tagging. Data are presented as mean ± SD from four biological replicates D Schematic representation of PRDM3 and corresponding protein fragments generated by IVTT. E NanoBit assay testing the interaction of PRDM3-SmBit protein fragments with the PAIRED domain of PAX8. LgBit-PRD/PRD-LgBit, N-terminal/C-terminal tagging. Data are presented as mean ± SD from two technical replicates from a representative experiment out of three independent experiments. F Overlay of the methyl region of 2D [ 13 C, 1 H]-HMQC spectra of uniformly 13 C, 15 N-labeled PAX8(9–135) in the absence (blue) and in the presence of unlabeled PRDM3 (2–345) at equimolar concentration (red). G Crosslinking-MS results from PAX8 (2–328) and PRDM3 (75–434). Intramolecular interactions are marked in purple and intermolecular interactions in green. Vertical blue bars inside protein diagrams represent the position of lysine residues and shaded regions represent domain boundaries. H CRISPR-Tiling screen data in OV56 (ovarian) and NCI-H1299 (lung) cell lines. Each dot represents a single sgRNA, and color coding is based on targeting a specific domain in PAX8 protein. Shaded vertical bar represents region enriched in intramolecular crosslinks from ( G ) corresponding to second helical portion of PAX8 DBD (called –RED). for Western blots and interaction measurements are provided as a Source Data file.

Article Snippet: Antibodies used are PAX8 (Cell Signaling, 59019, 1:10), PRDM3 (GenScript, U0869CG110-1, 1.5 μg).

Techniques: Generated, In Vitro, Labeling, Concentration Assay, CRISPR, Western Blot

Journal: Nature Communications

Article Title: PAX8 and MECOM are interaction partners driving ovarian cancer

doi: 10.1038/s41467-021-22708-w

Figure Lengend Snippet: A Venn diagram showing the overlap of ChIP-seq peaks of PAX8 and PRDM3 in ovarian cancer cells. *** P < 0.001 represents the statistical significance of the overlap between PAX8 and PRDM3 using Fisher’s exact test. B (Top) Sequence logo representation of the top motif identified by de novo motif finding in PAX8 + PRDM3 + sites and alignment to known PAX8 motif. (Bottom) Motif enrichment analysis for known PAX8 motif in PAX8 + PRDM3 + ChIP - seq peaks. C UCSC genome browser snapshot of the MANSC1 locus showing ChIP-seq tracks of PAX8 and PRDM3 in ovarian cells following shRNA-mediated knockdown of PAX8 or MECOM. shCTRL is a negative control. D Differential binding analyses of PAX8 (left) and PRDM3 (right) upon MECOM or PAX8 knockdown, respectively. MA plot represents the distribution of Log FC ( y -axis) and base mean coverage ( x -axis). Dots represent peaks with statistically significant differences (numbers indicated). E Representative FRAP images of PAX8-eGFP (green) and mCherry-PRDM3 (magenta) signal in the nucleus of U2OS cell. Arrow points to the bleached region with PAX8 hub. Scale bar = 10 µm. Average FRAP curves and quantification were generated by EasyFRAP-web tool. Mobile fraction of PAX8 in bleached region = 0.6; half-recovery time T 1/2 = 7 s; R 2 = 1. Mobile fraction of PRDM3 in bleached region = 1; half-recovery time T 1/2 = 15.2 s; R 2 = 1. F Expression heatmap of 58 genes from gene modules identified from RNA-seq experiments in five ovarian cancer cell lines upon PAX8 or MECOM knockdown. Log FC for the same 58 genes from in vivo xenografts studies is also plotted. for Western blots and qPCRs are provided as a Source Data file.

Article Snippet: Antibodies used are PAX8 (Cell Signaling, 59019, 1:10), PRDM3 (GenScript, U0869CG110-1, 1.5 μg).

Techniques: ChIP-sequencing, Sequencing, shRNA, Knockdown, Negative Control, Binding Assay, Generated, Expressing, RNA Sequencing, In Vivo, Western Blot

Journal: Nature Communications

Article Title: PAX8 and MECOM are interaction partners driving ovarian cancer

doi: 10.1038/s41467-021-22708-w

Figure Lengend Snippet: A Barplot showing sensitivity to PAX8 or MECOM KO as per CRISPR screens reported in DepMap portal. Bars are color coded by MECOM expression. B , C Tumor volume measurements of NIH:OVCAR3 cells bearing shRNAs against PAX8 ( B ) or MECOM ( C ). Trt start = day of starting daily doxycycline treatment. * P < 0.01 and *** P < 0.0001 signify significantly and highly significant differences to the respective vehicle (two-sided t test post hoc) on the last treatment day. Data are presented as mean ± SEM from n > 5 mice cohorts. D Western blot analysis of tumors from ( B ) 1 week after treatment start. E Boxplot of z -score expression of PAX8–MECOM Signature (Sig. Score) in TCGA ovarian cases ( n = 608) binned according to MECOM (left) or PAX8 (right) expression quartiles. Boxplots represent median and first and third quartiles, and whiskers extend to 95th percentile. P values are based on two-sided Wilcoxon’s rank-sum test. F Kaplan–Meier curve of survival from TCGA ovarian and endometrial patients bearing high or low levels of Signature Score (top and bottom quartile, n = 746). P P value from log-rank test. for Western blots and qPCRs are provided as a Source Data file.

Article Snippet: Antibodies used are PAX8 (Cell Signaling, 59019, 1:10), PRDM3 (GenScript, U0869CG110-1, 1.5 μg).

Techniques: CRISPR, Expressing, Western Blot

Journal: Virchows Archiv

Article Title: Malignant teratoid tumor of the thyroid gland: an aggressive primitive multiphenotypic malignancy showing organotypical elements and frequent DICER1 alterations—is the term “thyroblastoma” more appropriate?

doi: 10.1007/s00428-020-02853-1

Figure Lengend Snippet: Representative images of case 3. a Biphasic (right) and epithelial/tubule-predominant areas were seen juxtaposed in this area, note centrally located cartilage island. b The epithelial component was composed of primitive tubules lined by basophilic columnar cells admixed with glomeruloid papillary structures. c Highly cellular sarcomatoid stroma with scattered intestinal-type tubule and glomeruloid structures are seen. d In some areas, fetal-type tubules lined by clear cells are evident. Expression of PAX8 ( e ) and TTF1 ( f ) is predominantly mutually exclusive. Primitive small cell stroma shows variable expression of TTF1 as well ( g ). Otherwise, the stroma was focally desmin-positive ( h ) and diffusely SALL4 positive ( i ), note that tubules inconsistently expressed SALL4 in i

Article Snippet: Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, AR, USA) and the following antibodies: thyroglobulin (Clone 2H11 + 6E1, RTU, Cell Marque, Rocklin, CA), calcitonin (Clone SP17, RTU, Cell Marque, Rocklin, CA), TTF1 (clone 8G7G3/1, dilution, 1:500, Zytomed Systems, Berlin, Germany), PAX8 (rabbit polyclonal, 1:50, Cell Marque), pankeratin (clone AE1/AE3, 1:40, Zytomed), p63 (clone SFI-6, 1:100, DCS), AFP (clone EP209, 1:150, Cell Marque), CD117 (clone EP10, 1:100, Quartett), beta-HCG (polyclonal, 1:3000, Dako), SALL4 (clone 6E3, 1:100, Zytomed), gylpican-3 (clone 1 g12, 1:200, Zytomed), OCT3/4 (clone N1NK, 1:100, Novocastra), D2–40 (clone D2–40, 1:50, Zytomed), PLAP (clone 8A9, 1:25, Dako), CD30 (clone Ber-H2, 1:40, Zytomed), NSE (clone BBS/NC/VI-H1, 1:300, Dako), CD56 (clone MRQ-42, 1:100, CELL MARQUE), TP53 (clone DO-7, 1:50, Dako), WT1 (clone 6F-H2, 1:50, Dako), WT1-c-terminus (polyclonal, 1:50, Santa Cruz), S100 (polyclonal, 1:2500, Dako), synaptophysin (clone SY38, 1:50, Dako), CD34 (clone BI-3C5, 1:200, Zytomed), desmin (clone D33, 1:250, Dako), myogenin (clone F5D, 1:50, Dako), CDX2 (clone CDX2–88, 1:100, DCS), CK20 (clone Ks20.8, 1:50, Dako), HepPar-1 (clone OCH1E5, 1:200, Dako), NUT (clone C52B1, 1:45, Cell Signaling), TLE1 (polyclonal, 1:200, Santa Cruz), SMARCB1/INI1 (clone MRQ-27, dilution, 1:50, Zytomed), SMARCA4 (anti-BRG1 antibody, clone EPNCIR111A, 1:100, Abcam; Cambridge, UK), Ki-67 (clone 30–9, RTU, Ventana, Tucson, Arizona), chromogranin A (Clone LK2H10, 1/300, Cell Marque, Rocklin, CA), CD99 (Clone O13, RTU, Cell Marque, Rocklin, CA), GFAP (Clone GFA, 1/1000, DakoPatts, Denmark), and HMB45 (Clone HMB-45, 1/300, Cell Marque, Rocklin, CA).

Techniques: Expressing

Journal: Virchows Archiv

Article Title: Malignant teratoid tumor of the thyroid gland: an aggressive primitive multiphenotypic malignancy showing organotypical elements and frequent DICER1 alterations—is the term “thyroblastoma” more appropriate?

doi: 10.1007/s00428-020-02853-1

Figure Lengend Snippet: Clinicopathological and molecular features of reported DICER1-related teratoid thyroid malignancies including current study ( n = 8)

Article Snippet: Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, AR, USA) and the following antibodies: thyroglobulin (Clone 2H11 + 6E1, RTU, Cell Marque, Rocklin, CA), calcitonin (Clone SP17, RTU, Cell Marque, Rocklin, CA), TTF1 (clone 8G7G3/1, dilution, 1:500, Zytomed Systems, Berlin, Germany), PAX8 (rabbit polyclonal, 1:50, Cell Marque), pankeratin (clone AE1/AE3, 1:40, Zytomed), p63 (clone SFI-6, 1:100, DCS), AFP (clone EP209, 1:150, Cell Marque), CD117 (clone EP10, 1:100, Quartett), beta-HCG (polyclonal, 1:3000, Dako), SALL4 (clone 6E3, 1:100, Zytomed), gylpican-3 (clone 1 g12, 1:200, Zytomed), OCT3/4 (clone N1NK, 1:100, Novocastra), D2–40 (clone D2–40, 1:50, Zytomed), PLAP (clone 8A9, 1:25, Dako), CD30 (clone Ber-H2, 1:40, Zytomed), NSE (clone BBS/NC/VI-H1, 1:300, Dako), CD56 (clone MRQ-42, 1:100, CELL MARQUE), TP53 (clone DO-7, 1:50, Dako), WT1 (clone 6F-H2, 1:50, Dako), WT1-c-terminus (polyclonal, 1:50, Santa Cruz), S100 (polyclonal, 1:2500, Dako), synaptophysin (clone SY38, 1:50, Dako), CD34 (clone BI-3C5, 1:200, Zytomed), desmin (clone D33, 1:250, Dako), myogenin (clone F5D, 1:50, Dako), CDX2 (clone CDX2–88, 1:100, DCS), CK20 (clone Ks20.8, 1:50, Dako), HepPar-1 (clone OCH1E5, 1:200, Dako), NUT (clone C52B1, 1:45, Cell Signaling), TLE1 (polyclonal, 1:200, Santa Cruz), SMARCB1/INI1 (clone MRQ-27, dilution, 1:50, Zytomed), SMARCA4 (anti-BRG1 antibody, clone EPNCIR111A, 1:100, Abcam; Cambridge, UK), Ki-67 (clone 30–9, RTU, Ventana, Tucson, Arizona), chromogranin A (Clone LK2H10, 1/300, Cell Marque, Rocklin, CA), CD99 (Clone O13, RTU, Cell Marque, Rocklin, CA), GFAP (Clone GFA, 1/1000, DakoPatts, Denmark), and HMB45 (Clone HMB-45, 1/300, Cell Marque, Rocklin, CA).

Techniques: Variant Assay, Imaging

Journal: Cell chemical biology

Article Title: Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation.

doi: 10.1016/j.chembiol.2021.11.003

Figure Lengend Snippet: Figure 2. Dose-dependent inhibition of Pax2-dependent activation by a trio of triazolo pyrimidine derivatives (A) Pax2-transfected PRS4-Luc cells cultured in increasing concentrations of BG inhibitors were used to generate 12-point dose-response curves in duplicate (DF = 2.0). The maximum and minimum concentrations tested were 50 and 0.02 mM, respectively (gray circles). Both positive and negative controls consisted of Pax2-transfected PRS4-Luc cells treated with DMSO (black squares) and PRS4-Luc cells lacking Pax2 and without compound or vehicle treatment (black di- amonds). The accompanying bar plot reports the fold reduction of luminescence in BG-treated relative to vehicle-treated Pax2-transfected PRS4-Luc cells. (B) Tabulation of IC50 values and FR at 1.56 mM for the BG inhibitor trio. The PubChem SID numbers are listed below the designated compound names. (C) Chemical structures of the BG trio of Pax2 inhibitors. (D) Inhibition of Pax2, Pax5 or Pax8 mediated activation of PRS4-Luc with increasing concentrations of BG-1. DF, dilution factor; FR, fold reduction; IC50, 50%-maximal inhibitory concentration; and vehicle, DMSO/D6. Three-parameter curve fitting was used to fit logistic/ sigmoidal curves to the dose-response data (Hill slope = 1) using GraphPad Prism 8. Error bars represent 1 standard deviation from the calculated mean data points.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Cadherin-6 Cho et al., 1998 N/A Rabbit Pax2 Dressler and Douglass, 1992 N/A Rabbit PTIP Lechner et al., (2000) N/A Rabbit Pax8 Proteintech Cat#10336-1-AP Rabbit Beta-Actin Sigma-Aldrich Cat#A2066 Rabbit phospho-Histone H3 (Ser10) Millipore Cat#06-570 Rabbit Histone H3 Abcam Cat#ab1791 Rabbit phospho-MAPK Family Sampler kit Cell Signaling Technologies Cat#9910T Rabbit MAPK Family Sampler kit Cell Signaling Technologies Cat#9926 Rabbit Histone H3K4me3 Abcam Cat#ab8580 RNA Polymerase II CTD Abcam Cat#ab5408 Chemicals, peptides, and recombinant proteins BG-1 (CCG-191845) ChemDiv/Molport PubChem ID 124738812 BG-2 (CCG-130202) ChemDiv/Molport 124682279 BG-3 (CCG-191087) ChemDiv/Molport 124738112 Critical commercial assays Steady-Glo Promega Cat#E2520 CellTiter-Glo Promega Cat#G7572 iTaq SYBR Green master mix Bio-Rad Cat#1725121 Experimental models: Cell lines PRS4-Luc (HEK293) Patel, et al. 2012 N/A BRE-Luc (HEK293) Bradford, et al. 2019 N/A Human Renal Carcinoma Gnarra and Dressler 1995 N/A Human Ovarian Carcinoma SKOV3 ATCC HTB-77 Mouse IMCD3 ATCC CRL-2123 Oligonucleotides See Table S1 N/A Recombinant DNA CMV-Pax2b mouse Grimley and Dressler, 2018 N/A CMV-Pax2C63Y mouse Grimley and Dressler, 2018 N/A CMV-Luc Grimley and Dressler, 2018 N/A Software and algorithms Instant JChem 17.20.0 ChemAxon, Boston, MA https://chemaxon.com/products/instant-jchem Prism 8.4.2 GraphPad Software, San Diego, CA https://www.graphpad.com/scientific-software/prism/ MScreen (Jacob et al., 2012) https://mscreen.lsi.umich.edu/ ImageJ 2.0.0 (Schindelin et al., 2012) https://imagej.net/software/fiji/

Techniques: Inhibition, Activation Assay, Transfection, Cell Culture, Concentration Assay, Standard Deviation

Journal: Cell chemical biology

Article Title: Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation.

doi: 10.1016/j.chembiol.2021.11.003

Figure Lengend Snippet: Figure 6. Pax2 inhibitors disrupt PTIP recruitment and H3K4 methylation at the AVPR2 promoter (A) Western blots for the indicated proteins after culture in 300 mM NaCl () or 500 mM NaCl (+) for 24 h, with either DMSO (CNT) or 5 mM BG-1 or BG-2 as indicated. Note the high-salt induction of Pax2 protein in IMCD cells, regardless of inhibitors. (B) RT-PCR measures AVPR2 expression levels in control or BG-treated cells with and without high salt (*p < 0.01, one-way ANOVA and Tukey’s test). (C–G) Chromatin immunoprecipitation from treated IMCD cells as indicated, using primers for Pax2 binding site at the AVPR2 promoter. Note that binding of Pax2 is unaffected by inhibitors (C), whereas Pax8 does not bind well (D). BG-1 and BG-2 prevent recruitment of PTIP (E), H3K4 methylation (F), and Pol II recruitment (G). All ChIP data (C–G) were from three experiments, with error bars representing 1 SD. *p < 0.01, unpaired Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Cadherin-6 Cho et al., 1998 N/A Rabbit Pax2 Dressler and Douglass, 1992 N/A Rabbit PTIP Lechner et al., (2000) N/A Rabbit Pax8 Proteintech Cat#10336-1-AP Rabbit Beta-Actin Sigma-Aldrich Cat#A2066 Rabbit phospho-Histone H3 (Ser10) Millipore Cat#06-570 Rabbit Histone H3 Abcam Cat#ab1791 Rabbit phospho-MAPK Family Sampler kit Cell Signaling Technologies Cat#9910T Rabbit MAPK Family Sampler kit Cell Signaling Technologies Cat#9926 Rabbit Histone H3K4me3 Abcam Cat#ab8580 RNA Polymerase II CTD Abcam Cat#ab5408 Chemicals, peptides, and recombinant proteins BG-1 (CCG-191845) ChemDiv/Molport PubChem ID 124738812 BG-2 (CCG-130202) ChemDiv/Molport 124682279 BG-3 (CCG-191087) ChemDiv/Molport 124738112 Critical commercial assays Steady-Glo Promega Cat#E2520 CellTiter-Glo Promega Cat#G7572 iTaq SYBR Green master mix Bio-Rad Cat#1725121 Experimental models: Cell lines PRS4-Luc (HEK293) Patel, et al. 2012 N/A BRE-Luc (HEK293) Bradford, et al. 2019 N/A Human Renal Carcinoma Gnarra and Dressler 1995 N/A Human Ovarian Carcinoma SKOV3 ATCC HTB-77 Mouse IMCD3 ATCC CRL-2123 Oligonucleotides See Table S1 N/A Recombinant DNA CMV-Pax2b mouse Grimley and Dressler, 2018 N/A CMV-Pax2C63Y mouse Grimley and Dressler, 2018 N/A CMV-Luc Grimley and Dressler, 2018 N/A Software and algorithms Instant JChem 17.20.0 ChemAxon, Boston, MA https://chemaxon.com/products/instant-jchem Prism 8.4.2 GraphPad Software, San Diego, CA https://www.graphpad.com/scientific-software/prism/ MScreen (Jacob et al., 2012) https://mscreen.lsi.umich.edu/ ImageJ 2.0.0 (Schindelin et al., 2012) https://imagej.net/software/fiji/

Techniques: Methylation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Chromatin Immunoprecipitation, Binding Assay

Journal: Cell chemical biology

Article Title: Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation.

doi: 10.1016/j.chembiol.2021.11.003

Figure Lengend Snippet: Figure 7. Pax2 inhibitors disrupt Pax8-dependent PTIP recruitment and H3K4 methylation at the Slc14a2 promoter (A) Western blot shows 24 h high-salt induction of Pax8 protein in IMCD cells, regardless of 5 mM BG-1 or BG-2 inhibitor. (B) RT-PCR measures UTA1 expression levels in control or BG-treated cells with and without high salt (*p < 0.01). (C) RT-PCR measures UTA3 expression levels in control or BG-treated cells with and without high salt (*p < 0.01). (D–G) Chromatin immunoprecipitation from treated IMCD cells as indicated, using primers for Pax8 binding site at the Slc14a2 promoter. Note that binding of Pax8 is unaffected by inhibitors (D), whereas BG-1 and BG-2 prevent recruitment of PTIP (E), H3K4 methylation (F), and Pol II recruitment (G). For RT-PCR (B and C), *p < 0.01, one-way ANOVA and Tukey’s test. The ChIP data (D–G) were from three experiments, with error bars representing 1 SD. *p < 0.01, unpaired Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Cadherin-6 Cho et al., 1998 N/A Rabbit Pax2 Dressler and Douglass, 1992 N/A Rabbit PTIP Lechner et al., (2000) N/A Rabbit Pax8 Proteintech Cat#10336-1-AP Rabbit Beta-Actin Sigma-Aldrich Cat#A2066 Rabbit phospho-Histone H3 (Ser10) Millipore Cat#06-570 Rabbit Histone H3 Abcam Cat#ab1791 Rabbit phospho-MAPK Family Sampler kit Cell Signaling Technologies Cat#9910T Rabbit MAPK Family Sampler kit Cell Signaling Technologies Cat#9926 Rabbit Histone H3K4me3 Abcam Cat#ab8580 RNA Polymerase II CTD Abcam Cat#ab5408 Chemicals, peptides, and recombinant proteins BG-1 (CCG-191845) ChemDiv/Molport PubChem ID 124738812 BG-2 (CCG-130202) ChemDiv/Molport 124682279 BG-3 (CCG-191087) ChemDiv/Molport 124738112 Critical commercial assays Steady-Glo Promega Cat#E2520 CellTiter-Glo Promega Cat#G7572 iTaq SYBR Green master mix Bio-Rad Cat#1725121 Experimental models: Cell lines PRS4-Luc (HEK293) Patel, et al. 2012 N/A BRE-Luc (HEK293) Bradford, et al. 2019 N/A Human Renal Carcinoma Gnarra and Dressler 1995 N/A Human Ovarian Carcinoma SKOV3 ATCC HTB-77 Mouse IMCD3 ATCC CRL-2123 Oligonucleotides See Table S1 N/A Recombinant DNA CMV-Pax2b mouse Grimley and Dressler, 2018 N/A CMV-Pax2C63Y mouse Grimley and Dressler, 2018 N/A CMV-Luc Grimley and Dressler, 2018 N/A Software and algorithms Instant JChem 17.20.0 ChemAxon, Boston, MA https://chemaxon.com/products/instant-jchem Prism 8.4.2 GraphPad Software, San Diego, CA https://www.graphpad.com/scientific-software/prism/ MScreen (Jacob et al., 2012) https://mscreen.lsi.umich.edu/ ImageJ 2.0.0 (Schindelin et al., 2012) https://imagej.net/software/fiji/

Techniques: Methylation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Chromatin Immunoprecipitation, Binding Assay