Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Schematic diagrams of HEY1, NCOA2, and HEY1–NCOA2 chimeric proteins and their intracellular localization, as well as the induced expression of FLAG‐tagged HEY1, NCOA2, and HEY1–NCOA2 in the iPSC‐MSCs cell models. (A) Schematic diagrams of HEY1, NCOA2, and HEY1–NCOA2 proteins. Dashed lines represent exon–exon borders. Only coding exons are presented. (B) The mEGFP‐HEY1 , mEGFP‐NCOA2 , and mEGFP‐HEY1–NCOA2 constructs as well as the empty vector were introduced into iPSC MSC cells transiently and photographed at 24 h. The first row shows the direct EGFP fluorescence, the second row shows Hoechst staining of DNA, and the third row shows the merged image indicating the intracellular localization. Bars, 20 μm. (C) Immunoblot using anti‐Flag antibody showed the induced expression of FLAG‐tagged HEY1–NCOA2, HEY1, and NCOA2 in stably transduced iPSC‐MSCs.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Expressing, Construct, Plasmid Preparation, Fluorescence, Staining, Western Blot, Stable Transfection

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1–NCOA2 fusion protein DNA‐binding pattern in the genome. (A) Genomic context of HEY1 and HEY1–NCOA2 binding peaks, with a breakdown into three groups: (1) Peaks that are overlapping between HEY1 and HEY1–NCOA2 fusion protein, (2) HEY1‐only binding peaks, and (3) HEY1–NCOA2‐only binding peaks. (B) Signal enrichment heatmap for the peaks specific for HEY1 only (blue bar), HEY1–NCOA2 only (red bar), and overlapping peaks (purple bar). (C) Normalized enrichment of HEY1‐binding targets, comparing HEY1‐only peaks and HEY1‐binding peaks overlapping with HEY1–NCOA2‐binding peaks. (D) E‐box sequences bound by the HEY1 and HEY1–NCOA2 fusion protein. Distribution of the distance between binding peaks and the closest E‐box motif(s); the distance equals 0 for the direct overlap between peak and motif, which fraction is visualized with pie plots.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Binding Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Gene expression profile associated with HEY1–NCOA2. (A) Heatmap showing row‐normalized expression level of genes differentially regulated by HEY1‐NCOA2 in comparison to wildtype HEY1 [MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+)]. (B) GSEA Enrichment plot of the expression of genes that were identified as downregulated by HEY1 in MSC‐HEY1(+) versus MSC‐HEY1 ctrl . The gene rank was based on the log2(fold‐change) of the expression of genes in MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1‐NCOA2 ctrl . (C) GSEA enrichment plot for the expression of genes of which their promoters had binding peaks of both HEY1 and HEY1–NCOA2. The gene rank was based on the log2(fold‐change) of the gene expression in MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+). (D) Boxplots visualizing the enrichment of the 674 HEY1–NCOA2 direct‐target‐and‐transactivating genes identified using the iPSC‐MSCs cell models in sarcoma tumor samples. The enrichment was visualized across patients' samples from a total of eight sarcoma entities. GSEA, Gene Set Enrichment Analysis; NES, Normalized Enrichment Score; FDR, False Discovery Rate; MCS, Mesenchymal Chondrosarcoma; GIST, Gastrointestinal Stromal tumor; RHB, Rhabdomyosarcoma; IFS, Infantile Fibrosarcoma; ASPS, Alveolar Soft Part Sarcoma; SS, Synovial Sarcoma; DSRCT, Desmoplastic Small Round Cell Tumors; EWS, Ewing's Sarcoma.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Gene Expression, Expressing, Comparison, Binding Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Functional pathways enriched in HEY1–NCOA2 upregulated genes. (A) Bar plot showing the −log10(FDR) of the top‐10 enriched KEGG pathways, computed for 1,563 HEY1–NCOA2 genes upregulated by HEY1–NCOA2 as compared with HEY1 [MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+)]. (B) Heatmap summarizing the GSEA (Gene Set Enrichment Analysis) of selected gene sets. Each row represents one gene set. Row #4–#8, gene signatures were selected based on the KEGG pathway enrichment analysis. The values in the heatmap indicate the −log10 of p value, multiplied by −1 if the NES score was indicating the enrichment biased toward the negative phenotype. (C) GSEA enrichment plot of MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1‐NCOA2 ctrl and MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+). The gene set ‘Chondrocyte Differentiation’ is from Gene Ontology (GO). The false discovery rate (FDR), nominal p value and normalized enrichment score were calculated by GSEA.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Functional Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1–NCOA2 target gene expression validation. (A) RT‐qPCR validation of genes regulation by inducible HEY1, NCOA2, or HEY1–NCOA2 expression in iPSC MSC. The graph represents the fold change between the induced and uninduced condition. (B) Immunoblotting was performed to further confirm the expression of BCL2, CCND1, and HES1 after HEY1–NCOA2 induction in stably transduced iPSC‐MSCs. GAPDH was used as a loading control.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Targeted Gene Expression, Biomarker Discovery, Quantitative RT-PCR, Expressing, Western Blot, Stable Transfection, Control

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1‐NCOA2 significantly increases cell proliferation in iPSC‐MSC. (A) 1 × 10 5 of FACS sorted GFP+ iPSC‐MSCs were seeded with or without doxycycline (50 ng/ml). Cell numbers were counted on days 6 and 12; the graph shows the mean ± SD ( n = 3). (B) GFP+ iPSC‐MSCs were cultured with or without doxycycline (50 ng/ml) for 7 days and labeled with 10 μM of EdU for 3 h. Cells were then harvested, fixed, and stained with fluorescent dye 647 picolyl azide. EdU was detected by flow cytometry and the percentage of cells in S‐phase was analyzed using FlowJo software. The graph shows the mean ± SD ( n = 2). (C) FACS‐sorted GFP+ iPSC‐MSCs were cultured as in panel A except without addition of rh‐FGF basic and rh‐IGF‐1 to the culture medium. Cell numbers were counted on day 7; the graph shows the mean ± SD ( n = 3).

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Cell Culture, Labeling, Staining, Flow Cytometry, Software

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

doi: 10.1007/s10495-012-0713-6

Figure Lengend Snippet: Fig. 4 Selective roles of PI3-K C isoforms in the maintenance of HIEC cell survival. a Representative (n C 4) siRNA-validating WB analyses of the expression of 55c, p85b, and p110a, in HIEC cells transfected with siCNS (control), sip55c, sip85b, or sip110a, using isoform-specific antibodies. b HIEC cells were transfected with siCNS (control), sip110a, sip110b, sip85a, sip85b, sip55c, or a combination of sip55c ? sip85b. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. Statistically significant (0.0001 B P B 0.001) differences between treated and control cultures are indicated by (*); statistically significant (0.0005 B P B 0.005) differences between sip85b, or sip55c, with sip110a are indicated by (#); statistically significant (0.0005 B P B 0.005) differences between sip85b, or sip55c, with the combination sip55c ? sip85b are indicated by (**); statistically significant (0.001 B P B 0.01) differences between sip55c and sip85b are indicated by (##)

Article Snippet: Small interference RNA (siRNA)-mediated expression silencing assays siRNAs specifically directed against the mRNAs of p110a (sip110a), p110b (sip110b), p85a (sip85a), p85b (sip85b) or p55c (sip55c) were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Transfection, Control, Activity Assay, Comparison

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

doi: 10.1007/s10495-012-0713-6

Figure Lengend Snippet: Fig. 7 Selective engagement of PI3-K isoform complexes by integrin/Fak/Src-mediated signaling in HIEC cells. a Representative (n C 3) WB analyses of the IP of Fak for the verification of association by co-IP of p110a, p85b, and p55c, as well as verification of Fak activation, from HIEC cell control cultures or cultures treated with PF573228, PP2 or kept in suspension in polyHEMA-coated dishes (suspension). Specific antibodies for each PI3-K isoform probed, as well as for pY397p125Fak and p125Fak, were used. b Representative (n C 3) WB analyses of the IP of Src for the verification of association by co-IP of p85b and p55c, as well as verification of Fak-Src interactions and Src activation, from HIEC cell control cultures or cultures treated with PF573228, PP2 or kept in suspension in polyHEMA-coated dishes (suspension). Specific anti- bodies for each PI3-K isoform probed, as well as for pY576/577p125Fak, pY418p60Src and p60Src, were used

Article Snippet: Small interference RNA (siRNA)-mediated expression silencing assays siRNAs specifically directed against the mRNAs of p110a (sip110a), p110b (sip110b), p85a (sip85a), p85b (sip85b) or p55c (sip55c) were purchased from OriGene (Rockville, MD).

Techniques: Co-Immunoprecipitation Assay, Activation Assay, Control, Suspension

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

doi: 10.1007/s10495-012-0713-6

Figure Lengend Snippet: Fig. 8 PI3-K isoforms and isoform complexes are selectively expressed, perform distinct roles in cell survival, and are selectively engaged by b1 integrin/Fak/Src-mediated signaling for the suppres- sion of anoikis, in HIEC cells. Schematic drawing, which summarizes the results of the present study. p110a/p85b and p110a/p55c are the largely predominant PI3-K isoform complexes in HIEC cells, whereas the individual isoforms p85a and p110b are expressed weakly, and p110c and p110d are not expressed. Only the p110a/p85b and p110a/ p55c complexes perform the critical functions of Akt-1 activation and subsequent maintenance of HIEC cell survival. However, the contributions of p110a/p55c in Akt-1 activation and cell survival are significantly greater than those of p110a/p85b. Furthermore, p110a/p85b and p110a/p55c are both engaged by b1 integrin/Fak/Src signaling in the suppression of anoikis; nevertheless, the engagement of p110a/p85b is primarily Src-dependent, whereas that of p110a/ p55c is primarily Fak-dependent (but Src-independent)

Article Snippet: Small interference RNA (siRNA)-mediated expression silencing assays siRNAs specifically directed against the mRNAs of p110a (sip110a), p110b (sip110b), p85a (sip85a), p85b (sip85b) or p55c (sip55c) were purchased from OriGene (Rockville, MD).

Techniques: Activation Assay