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: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: C18-3OH modulates ABCA1 expression and cholesterol efflux in foam cells via PAX-8 upregulation. (A) RT-qPCR analysis of PAX-8 mRNA expression in foam cells treated with C18-3OH. (B) Western blot analysis of PAX-8 protein expression in foam cells treated with C18-3OH. ** P < 0.01 vs. control, n = 3. (C) Western blot detection of PAX-8 and ABCA1 protein expression in foam cells following PAX-8 knockdown. (D) Quantitative analysis of PAX-8 protein expression (Panel C). (E) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after PAX-8 knockdown. (F) Quantitative analysis of ABCA1 protein expression (Panel C). (G) Effects of C18-3OH and siPAX-8 on cholesterol efflux in foam cells. * P < 0.05, ** P < 0.01 vs. siNC + DMSO; ## P < 0.01 vs. siNC + C18-3OH, n = 3. (H) NBD-cholesterol fluorescence intensity in foam cells; scale bar = 150 μm.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Knockdown, Fluorescence

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: PAX-8 binds to and regulates ABCA1 expression. (A) Predicted binding sites between PAX-8 and ABCA1 in humans. (B) Predicted binding sites between PAX-8 and ABCA1 in mice. (C) Western blot analysis of PAX-8 and ABCA1 protein expression in foam cells following PAX-8 overexpression. (D) Quantitative analysis of PAX-8 protein expression (Panel C). (E) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after PAX-8 overexpression. (F) Quantitative analysis of ABCA1 protein expression (Panel C). ** P < 0.01 vs. LV-NC, n = 3. (G) ChIP-Seq identification of PAX-8 binding to the ABCA1 locus. Genomic tracks showing PAX-8 binding to the ABCA1 locus in the Input (negative control, top) and ChIP (PAX-8-enriched, bottom) groups. The y-axis indicates relative enrichment of ChIP-Seq signals. The x-axis represents genomic coordinates. (H) ChIP-qPCR validation of PAX-8 targeting regulation of ABCA1. ** P < 0.01 vs. Input, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Binding Assay, Western Blot, Over Expression, Quantitative RT-PCR, ChIP-sequencing, Negative Control, ChIP-qPCR, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on m 6 A modification of PAX-8 mRNA in foam cells. (A) Predicted score distribution of the human PAX-8 gene query sequence. (B) Predicted score distribution of the murine PAX-8 gene query sequence. (C) Partitioned statistical plot of predicted scores for the human PAX-8 gene query sequence. (D) Partitioned statistical plot of predicted scores for the murine PAX-8 gene query sequence. (E) MeRIP-qPCR analysis of the effect of C18-3OH on m 6 A modification of PAX-8 mRNA in foam cells. ** P < 0.01 vs. control, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Modification, Sequencing, Control

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on ALKBH5 expression in foam cells. (A) Bioinformatic analysis of methylation-associated proteins regulating PAX-8 in THP-1 cells. (B) RT-qPCR analysis of ALKBH5 mRNA expression in foam cells treated with C18-3OH. (C) Western blot analysis of ALKBH5 protein expression in foam cells treated with C18-3OH. (D) RT-qPCR analysis of ELAVL1 mRNA expression in foam cells treated with C18-3OH. (E) Western blot analysis of ELAVL1 protein expression in foam cells treated with C18-3OH. * P < 0.05, ** P < 0.01 vs. control, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: ALKBH5 mediates C18-3OH regulation of PAX-8 and ABCA1 expression, PAX-8 mRNA m 6 A modification, and cholesterol efflux. (A) Western blot analysis of ALKBH5 expression in foam cells following lentiviral transfection for ALKBH5 overexpression. ** P < 0.01 vs. control, n = 3. (B) Western blot analysis of PAX-8 and ABCA1 protein expression in foam cells following ALKBH5 overexpression. (C) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after ALKBH5 overexpression. (D) Quantitative analysis of ABCA1 protein expression (Panel B). (E) RT-qPCR analysis of PAX-8 mRNA expression in foam cells after ALKBH5 overexpression. (F) Quantitative analysis of PAX-8 protein expression (Panel B). (G) MeRIP-qPCR analysis of PAX-8 mRNA m6A modification in foam cells following ALKBH5 overexpression. (H) Effect of C18-3OH and LV-ALKBH5 on cholesterol efflux in foam cells. (I) NBD-cholesterol fluorescence intensity in foam cells; scale bar = 150 μm. * P < 0.05, ** P < 0.01 vs. LV-NC + DMSO; ## P < 0.01 vs. LV-NC + C18-3OH, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Modification, Western Blot, Transfection, Over Expression, Control, Quantitative RT-PCR, Fluorescence

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on ABCA1, PAX-8, ALKBH5 expression, and PAX-8 mRNA m 6 A methylation in Aortas of apoE −/− Mice. (A) Western blot analysis of ABCA1, PAX-8, and ALKBH5 protein expression in aortas of HFD-fed apoE −/− mice treated with C18-3OH and/or ALKBH5 overexpression. (B) RT-qPCR analysis of ABCA1 mRNA expression in aortas of apoE −/− mice. (C) RT-qPCR analysis of PAX-8 mRNA expression in aortas of apoE −/− mice. (D) RT-qPCR analysis of ALKBH5 mRNA expression in aortas of apoE −/− mice. (E) Quantitative analysis of ABCA1 protein expression (Panel A). (F) Quantitative analysis of PAX-8 protein expression (Panel A). (G) Quantitative analysis of ALKBH5 protein expression (Panel A). (H) MeRIP-qPCR analysis of PAX-8 mRNA m 6 A methylation levels in aortas of apoE −/− mice across experimental groups. ** P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. C18-3OH + AAV-NC. n=6.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Methylation, Western Blot, Over Expression, Quantitative RT-PCR, Control

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: Protein samples were loaded on SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with the following antibodies: GAPDH (Cell Signaling, 8884; 1:1000 dilution), HA (BioLegend, 901501; 1:1000 dilution), LgBit (R&D systems, MAB10026, 1:1000 dilution), MECOM (Cell Signaling, 2593; 1:1000 dilution), PAX8 (Cell Signaling, 59019; 1:1000 dilution), PRDM3 (GenScript, U0869CG110-1; 1:10,000 dilution), VINCULIN (Sigma, V9131; 1:400 dilution), and HRP‐anti‐rabbit and HRP-anti-mouse (Cell Signaling).

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: Protein samples were loaded on SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with the following antibodies: GAPDH (Cell Signaling, 8884; 1:1000 dilution), HA (BioLegend, 901501; 1:1000 dilution), LgBit (R&D systems, MAB10026, 1:1000 dilution), MECOM (Cell Signaling, 2593; 1:1000 dilution), PAX8 (Cell Signaling, 59019; 1:1000 dilution), PRDM3 (GenScript, U0869CG110-1; 1:10,000 dilution), VINCULIN (Sigma, V9131; 1:400 dilution), and HRP‐anti‐rabbit and HRP-anti-mouse (Cell Signaling).

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: Protein samples were loaded on SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with the following antibodies: GAPDH (Cell Signaling, 8884; 1:1000 dilution), HA (BioLegend, 901501; 1:1000 dilution), LgBit (R&D systems, MAB10026, 1:1000 dilution), MECOM (Cell Signaling, 2593; 1:1000 dilution), PAX8 (Cell Signaling, 59019; 1:1000 dilution), PRDM3 (GenScript, U0869CG110-1; 1:10,000 dilution), VINCULIN (Sigma, V9131; 1:400 dilution), and HRP‐anti‐rabbit and HRP-anti-mouse (Cell Signaling).

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: Protein samples were loaded on SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with the following antibodies: GAPDH (Cell Signaling, 8884; 1:1000 dilution), HA (BioLegend, 901501; 1:1000 dilution), LgBit (R&D systems, MAB10026, 1:1000 dilution), MECOM (Cell Signaling, 2593; 1:1000 dilution), PAX8 (Cell Signaling, 59019; 1:1000 dilution), PRDM3 (GenScript, U0869CG110-1; 1:10,000 dilution), VINCULIN (Sigma, V9131; 1:400 dilution), and HRP‐anti‐rabbit and HRP-anti-mouse (Cell Signaling).

Techniques: CRISPR, Expressing, Western Blot

Journal: Science signaling

Article Title: Repurposing colforsin daropate to treat MYC-driven high-grade serous ovarian carcinomas.

doi: 10.1126/scisignal.ado8303

Figure Lengend Snippet: Fig. 4. CF inhibits HGSOC tumor growth in vivo. (A) Diagram depicting dosing schedule for HEYA8 subcutaneous tumor formation experiment. (B) Representative im- ages of subcutaneous HEYA8 tumors harvested from mice treated with either vehicle, CF, Cis, or the 1:1 CF-Cis combination. (C) Graph of tumor kinetics for mice treated with either vehicle, CF, Cis, or the 1:1 CF-Cis combination. Error bars represent ± SEM. (D) Graph of Bliss and HSA in vivo synergy CIs for CF-Cis–treated HEYA8 tumors. A CI greater than 0 represents synergy between the two treatment groups, a CI equal to 0 represents independent effects for the two treatment groups, and a CI less than 0 represents antagonism between the two treatment groups. (E) Representative micrographs of hematoxylin and eosin (H&E), PAX8, and Ki-67 staining of vehicle-, CF-, Cis-, and 1:1 CF-Cis combination–treated tumors. Scale bars for images are 25 μm. (F) Graph showing % positive area for Ki-67 in vehicle-, CF-, Cis-, or 1:1 CF-Cis combination– treated tumors. (G) Graph showing final tumor burden for vehicle-, CF-, Cis-, or 1:1 CF-Cis combination–treated tumors. (H) Diagram depicting dosing schedule for OVCAR4-Luc intraperitoneal (IP) tumor formation experiment. (I) Representative bioluminescence images of intraperitoneal tumor kinetics at days 0 to 168 for mice treated with either vehicle, CF, Cis, or the 1:1 CF-Cis combination. Red lines indicate where images have been cropped together to show N = 5 representative mice from different cages. Magenta lines indicate where images have been cropped together to remove empty slots present during imaging. (J) Graph of intraperitoneal tumor ki- netics for mice treated with either vehicle, CF, Cis, or the 1:1 CF-Cis combination. Error bars represent ± SEM. (K) Graph of Bliss and HSA in vivo synergy CIs for CF-Cis– treated OVCAR4-Luc tumors. (L) Kaplan-Meier survival curves for OVCAR4-Luc mice treated with either vehicle, CF, Cis, or the 1:1 CF-Cis combination. Statistical comparisons between treatment groups are shown next to the legend and were performed using the log-rank test. Dashed red line indicates the time point where the study was ter- minated and all surviving mice were euthanized. All data [(A) to (L)] are representative of at least N = 3 biological replicates. For all in vivo experiments, N ≥ 5 mice for each treatment group. Statistical comparisons of two groups were performed using Student’s t test, and comparisons of three or more groups were performed using one-way ANOVA with Tukey multiple testing correction unless otherwise stated.

Article Snippet: Immunohistochemical staining was performed using a 1:500 dilution of antibodies to PAX8 (Novus, NBP1- 32440), Ki- 67 (CST, 12202), and MYC (CST, 5605).

Techniques: In Vivo, Staining, Imaging