Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: (A) Unsupervised hierarchical clustering of MECOM , PAX8 , SOX17 and WT1 mRNA expression in the pan-normal GTEx dataset. TCGA data was clustered based on the 5 main clusters of TF expression from GTEx. MTF low = Cluster of tissues that did not or lowly expressed MTFs, MTF high = cluster of tissues that moderately or highly expressed all four MTFs, M high = Cluster of tissues that highly expressed MECOM, M&P high = cluster of tissues that highly expressed both MECOM and PAX8, P high = cluster of tissues that expresses PAX8 and S high = cluster of tissues that expresses SOX17). (B) A boxplot of the average Pearson correlation values of MECOM, PAX8, SOX17 and WT1. Ranked from highest average Pearson correlation value to lowest. (C) A boxplot representing the mean positivity rate of MECOM, PAX8, SOX17 and WT1 expression in each histotype. In B and C, the limits of the boxes represent the interquartile range, and the limits of error bars represent the minimum and maximum value without outliers (D) Ratio of samples with number of co-stained TFs based on a 0.1 positivity rate threshold.

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: Landscapes of active chromatin and chromatin loops (based on Hi-C maps) in FTSECs and HGSCs. (A) MECOM, (B) PAX8, (C) SOX17 and (D) WT1 .

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Hi-C

Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: (A) TF knock-down followed by colony formation assays stained with crystal violet. Representative wells are shown. (B) Barplots representing the quantification of crystal violet staining in . Error bars represent biological replicates. (C) Dose response curves for FT246, FT282, KURAMOCHI and OVCAR4 cells treated with THZ1, THZ531 and JQ1. Error bars represent standard deviation of mean cell survival values from biological replicates. (D) RT-qPCR quantification of MECOM , PAX8 , SOX17 and WT1 expression upon THZ1 and THZ531 treatment in FT282 and OVCAR4 cells. Data for MECOM , PAX8 and SOX17 expression upon THZ1 and THZ531 treatment of OVCAR4 cells are reproduced from .

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Knockdown, Staining, Standard Deviation, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: (A) MECOM, PAX8, SOX17 and WT1 co-occupies its own and others genomic loci. ( B) MECOM, PAX8, SOX17 and WT1 co-occupy active enhancer regions across the genome. CPM-normalized CUT&RUN reads were centered on 3 kilobase windows of FTSEC or HGSC-specific PAX8 peaks. Rows are the same across feature. (C) Metagene plot, MECOM, PAX8, SOX17, WT1, H3K27ac and H3K27me3 signal centered on FT of HGSC-specific PAX8 peaks. (D) Set analysis of CUT&RUN peaks from representative MECOM, PAX8, SOX17 and WT1 samples in FT282 and KURAMOCHI. (E) Chromatin state of TF peaks categorized by number of TF overlaps. (F) MECOM, PAX8, SOX17 and WT1 co-regulation based on TF knock-down followed by RNA-seq and differential expression analysis with DESEQ2. (G) Node and edge plot representing co-regulation of each TF based on gene expression measured by RNA-seq after TF knock-down.

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Knockdown, RNA Sequencing, Quantitative Proteomics, Gene Expression

Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: (A) Set analysis of TF binding sites that were common or specific to FT282 or KURAMOCHI. (B) MECOM, PAX8, SOX17 and WT1 co-occupies regions that were bound in a common or context-specific manner. CPM-normalized CUT&RUN reads were centered on 3 kilobase windows of PAX8 peak start and stop positions. Rows are the same across samples. (C) Ratio of chromatin states associated with TF binding regions categorized by cellular context. (D) Ratio of FT282 and KURAMOCHI specific enhancers bound by one, two, three or four TFs. (E) BHLHE41 and PBX1 loci displaying the co-localization of MECOM, PAX8, SOX17 and WT1 at a KURAMOCHI specific super-enhancer.

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development

doi: 10.1101/2023.04.11.536378

Figure Lengend Snippet: (A) Log 2 fold-change of 28,158 genes following normalization informed by ERCC spike-in RNA content, (B) Number of differentially expressed genes for each TF knock-down based on absolute log 2 fold-change ≥ 0.5. (C) Schematic to integrate differential expression, TF binding and topologically associated domain (TAD) maps. (D) Alluvial plot displaying the status of high confidence differentially expressed genes following TF depletion in FTSECs and HGSCs. (E) Heatmap and pathway analysis of genes displayed in D. (F) BRCA1 locus highlighting H3K27ac signal and TF binding at the BRCA1 promoter. (G) Log 2 fold change of BRCA1 expression following MECOM, PAX8, SOX17 and WT1 knock-down relative to scrambled controls. (H) Chromatin landscape of RUNX3 locus in FTSECs and HGSCs. (I) Log 2 fold change of RUNX3 expression following MECOM, PAX8, SOX17 and WT1 knock-down relative to scrambled controls (RNA-seq).

Article Snippet: Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611).

Techniques: Knockdown, Quantitative Proteomics, Binding Assay, Expressing, RNA Sequencing

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Zengmian Yiliu formula suppresses cell cycle in immune-rich ovarian cancer patient-derived organoids.

doi: 10.1016/j.phymed.2025.156721

Figure Lengend Snippet: Fig. 2. Ovarian cancer organoid characterization. (A) H&E staining of organoids from six patients (OV075, OV076, OV077, OV078, OV080, and OV082), showcasing cellular arrangement and tissue structure. (B) PAX8 staining confirms the organoids’ ovarian epithelial origin. (C) Ki67 staining assesses the organoids’ proliferative activity. Scale Bar: 20 µm.

Article Snippet: For IHC, after deparaffinization and antigen retrieval, the sections were incubated with primary antibodies against Ki67 (MA5-14520, ThermoFisher) and PAX8 (10336-1-AP, Proteintech) at a dilution of 1:1000 overnight at 4◦C.

Techniques: Staining, Activity Assay

Journal: Oncogene

Article Title: HBx regulates transcription factor PAX8 stabilization to promote the progression of hepatocellular carcinoma.

doi: 10.1038/s41388-019-0907-2

Figure Lengend Snippet: Fig. 4 Skp2 mediate PAX8 protein ubiquitination and degradation. a, b Western blot and qRT-PCR analysis of PAX8 expression in HepG2 cells following transfected with the indicated dose of Skp2-specific siRNA or control siRNA. c, d Western blot analysis of PAX8 and His-Skp2 in HepG2 or HEK293T cells following inducing variable levels of His-Skp2. +:2 ug, ++:3 ug, +++:4 ug. e Treatment with MG132 mitigates the Skp2- decreased PAX8 protein in HepG2 cells. f Induction of His-Skp2 expression reduces PAX8 protein ubiquitination and degradation in HepG2 cells after treatment with CHX. g Skp2 silencing mitigates PAX8 protein ubiquitination and degradation in HEK293T cells following cotransfection with the indicated plasmid and siRNA. h Induction of His-Skp2 expression enhances PAX8 protein ubiquitination and degradation in HEK293T cells. i, j Skp2, but not its Skp2ΔF mutant promotes PAX8 protein ubiquitination and degradation in HepG2 and HEK293T cells. Data are representative images or expressed as the mean or mean ± SD of each group from three separate experiments. *P < 0.05, **P < 0.01

Article Snippet: After being blocked with 5% fat-free dry milk in TBST, the membranes were incubated with antibodies against PAX8 (Novus, NBP1-32440), HBx (XIAMEN INNOVAX BIOTECH), Rb (#9313), phosphor-Rb (#D59B7), P21 (#2947), E2F1 (sc251), p53 (sc-126), CDK2 (#78B2), and GAPDH (60004-1- Ig, Santa Cruz Biotechnology or Proteintech).

Techniques: Ubiquitin Proteomics, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, Cotransfection, Plasmid Preparation, Mutagenesis

Journal: Oncogene

Article Title: HBx regulates transcription factor PAX8 stabilization to promote the progression of hepatocellular carcinoma.

doi: 10.1038/s41388-019-0907-2

Figure Lengend Snippet: Fig. 6 HBx inhibits the Skp2- mediated PAX8 ubiquitination. a, b Immunoprecipitation indicates that HBx directly interacts with Skp2 in HepG2 and HEK293T cells. c Immunofluorescent confocal microscopy reveals that HBx and Skp2 are colocalized in the perinuclear of HepG2 cells. d HBx interacts with His-Skp2, but not His-Skp2ΔLRR, in HEK293T cells. e, f HBx inhibits the Skp2-reduced PAX8 in HEK293T and HepG2 cells. g HBx mitigates the Skp2- mediated PAX* ubiquitination and degradation in HEK293T cells. h Western blot analysis of cytosol and nuclear PAX8 in HBx+ HepG2-HBx and HepG2.2.15 cells. Data are representative images of each group of cells from three separate experiments

Article Snippet: After being blocked with 5% fat-free dry milk in TBST, the membranes were incubated with antibodies against PAX8 (Novus, NBP1-32440), HBx (XIAMEN INNOVAX BIOTECH), Rb (#9313), phosphor-Rb (#D59B7), P21 (#2947), E2F1 (sc251), p53 (sc-126), CDK2 (#78B2), and GAPDH (60004-1- Ig, Santa Cruz Biotechnology or Proteintech).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Confocal Microscopy, Western Blot