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Boster Bio anti fli1
Anti Fli1, supplied by Boster Bio, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+fli1/pmc11516120-323-23-24?v=Boster+Bio
Average 93 stars, based on 1 article reviews

anti fli1 - by Bioz Stars, 2026-08

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A EWS::FLI1 and CD99 expression in heMSCs at 48 h after infection and in A673 cells. Actin, loading control. At the bottom, densitometric quantification of western blot signals normalized to Actin intensity. RT-qPCR data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. B Western blot detection of proteins of the p53-p21-RB1 axis in EF-heMSCs ( n = 2 independent experiments). Actin, loading control. C RT-qPCR to detect the expression of some of the induced and repressed oncogene targets in heMSC-1 cells at 48 h after EWS::FLI1 infection ( n = 2 independent experiments performed in triplicate). Data express mean ± s.d. Statistics performed by two-tailed unpaired t -test. D Gene-concept networks of the top 5 significantly enriched terms in EF-heMSCs vs . control heMSCs. Data was analyzed as indicated in Fig. . E RT-qPCR to determine the expression of 15 of the 30 top genes most potently induced by EWS::FLI1 in heMSC-1 cells at 48 h. Data from three independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. F Expression values obtained from DepMap of the genes shown in ( E ) in Ewing sarcoma and other cancer cell lines (log2(TPM + 1)). A two-tailed Mann–Whitney U test was performed. G GSEA of EF-heMSCs transcriptomes in EWS::FLI1 signatures (EWS::FLI1 expression in UET-13 mesenchymal progenitors , rhabdomyosarcoma RD cells , and hMSCs ). H GSEA of EF-heMSCs transcriptomes in Ewing sarcoma signatures , . I , J Heatmap and PCA representation of the unsupervised clustering analysis of gene expression signatures in heSCs, hMSCs, and Ewing sarcoma samples. K GSEA of EF-heMSCs transcriptomes in the 400 most expressed and under-represented genes identified by the unsupervised clustering analysis of Ewing sarcomas. In ( G , H , K ), a two-sided test was performed with GSEA based on limma-derived statistics (−log( p value) × signFC). Running score plots show the cumulative enrichment of the gene set across the ranked gene list. The peak of the curve indicates the maximum enrichment score (ES). P values are adjusted for multiple testing using the Benjamini–Hochberg (FDR < 0.05).

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A EWS::FLI1 and CD99 expression in heMSCs at 48 h after infection and in A673 cells. Actin, loading control. At the bottom, densitometric quantification of western blot signals normalized to Actin intensity. RT-qPCR data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. B Western blot detection of proteins of the p53-p21-RB1 axis in EF-heMSCs ( n = 2 independent experiments). Actin, loading control. C RT-qPCR to detect the expression of some of the induced and repressed oncogene targets in heMSC-1 cells at 48 h after EWS::FLI1 infection ( n = 2 independent experiments performed in triplicate). Data express mean ± s.d. Statistics performed by two-tailed unpaired t -test. D Gene-concept networks of the top 5 significantly enriched terms in EF-heMSCs vs . control heMSCs. Data was analyzed as indicated in Fig. . E RT-qPCR to determine the expression of 15 of the 30 top genes most potently induced by EWS::FLI1 in heMSC-1 cells at 48 h. Data from three independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. F Expression values obtained from DepMap of the genes shown in ( E ) in Ewing sarcoma and other cancer cell lines (log2(TPM + 1)). A two-tailed Mann–Whitney U test was performed. G GSEA of EF-heMSCs transcriptomes in EWS::FLI1 signatures (EWS::FLI1 expression in UET-13 mesenchymal progenitors , rhabdomyosarcoma RD cells , and hMSCs ). H GSEA of EF-heMSCs transcriptomes in Ewing sarcoma signatures , . I , J Heatmap and PCA representation of the unsupervised clustering analysis of gene expression signatures in heSCs, hMSCs, and Ewing sarcoma samples. K GSEA of EF-heMSCs transcriptomes in the 400 most expressed and under-represented genes identified by the unsupervised clustering analysis of Ewing sarcomas. In ( G , H , K ), a two-sided test was performed with GSEA based on limma-derived statistics (−log( p value) × signFC). Running score plots show the cumulative enrichment of the gene set across the ranked gene list. The peak of the curve indicates the maximum enrichment score (ES). P values are adjusted for multiple testing using the Benjamini–Hochberg (FDR < 0.05).

Article Snippet: Antibodies used for these techniques were: FLI1, p53, p21, ATM, hHLA, PRKCB (Santa Cruz); CD99, hKu80(Cell Signaling); β-actin (Abcam); RB1 (BD Biosciences); human nucleus, BRCA1, pS1981ATM(Millipore); pS1423-BRCA1, ATR, pT1989-ATR, DNA-PKc, pS2056-DNA-PKcs (Abclonal); Flag (Merck Life Science); BCL11B (Biolegend) and ITM2A (Fisher Scientific).

Techniques: Expressing, Infection, Control, Western Blot, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Gene Expression, Derivative Assay

A Left, genomic annotation of oncogene-bound regions in heMSC-1 cells 48 h after infection with a Flag-tagged EWS::FLI1, identified by ChIP-seq performed with a Flag antibody. Right, overlapping of peaks. Peak calling using the input as control was performed with MACS2 . B Chromatin states associated with EWS::FLI1-bound peaks in heMSC-1 cells, performed with MACS2 tools using five core histone modification marks : H3K27me3 (Polycomb repression, ReprPC); H3K9me3 (heterochromatin regions, Het); H3K4me1 (enhancer regions, Enh); H3K4me3 (promoter regions, TssA); and H3K36me3 (transcribed regions, Tx). Statistical significance of the relative frequency of EWS::FLI1 peaks in each chromatin state was assessed using a two-sided Fisher’s exact test. Numbers in the bars indicate P values, odds ratios, and confidence intervals. Quiet (Quiescent/Low) chromatin state was excluded from this graph to better visualize the data. C Percentage of EWS::FLI1-binding sites upstream and downstream from the transcriptional start sites (TSS) of the nearest genes. D Identification of EWS::FLI1-binding motifs by MEME tools . E Overlap of genes associated with EWS::FLI1-binding peaks in heMSC and A673 cells. Bottom panel, annotations of EWS::FLI1 peaks in heMSCs corresponding to oncogene-bound genes. F Genome browser tracks depicting EWS::FLI1 binding to the PRKCB locus in heMSCs (top) and A673 cells (bottom) . In blue, heMSC-1 cells infected with EWS::FLI1; in gray, heMSC-1 cells infected with control supernatants. Scale, 0-23. Bottom left panel, validation of EWS::FLI1 binding to intron 7 of PRKCB in heMSCs, detected by ChIP-qPCR in EF-heMSC-1 cells. Values referred to the percentage of input and were normalized with respect to the control condition. ACAT1 , negative control. Bottom right panel, PRKCB induction is abolished after EWS::FLI1 knockdown. Data from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. G Sankey plots showing annotations of peaks corresponding to genes bound by EWS::FLI1 in both heMSC and A673 cells. In each of the plots, the transitions from distal intergenic (on the left), first intron (in the middle), and other introns (on the right) of the oncogene peaks in heMSCs to the peaks in A673 cells have been highlighted.

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Left, genomic annotation of oncogene-bound regions in heMSC-1 cells 48 h after infection with a Flag-tagged EWS::FLI1, identified by ChIP-seq performed with a Flag antibody. Right, overlapping of peaks. Peak calling using the input as control was performed with MACS2 . B Chromatin states associated with EWS::FLI1-bound peaks in heMSC-1 cells, performed with MACS2 tools using five core histone modification marks : H3K27me3 (Polycomb repression, ReprPC); H3K9me3 (heterochromatin regions, Het); H3K4me1 (enhancer regions, Enh); H3K4me3 (promoter regions, TssA); and H3K36me3 (transcribed regions, Tx). Statistical significance of the relative frequency of EWS::FLI1 peaks in each chromatin state was assessed using a two-sided Fisher’s exact test. Numbers in the bars indicate P values, odds ratios, and confidence intervals. Quiet (Quiescent/Low) chromatin state was excluded from this graph to better visualize the data. C Percentage of EWS::FLI1-binding sites upstream and downstream from the transcriptional start sites (TSS) of the nearest genes. D Identification of EWS::FLI1-binding motifs by MEME tools . E Overlap of genes associated with EWS::FLI1-binding peaks in heMSC and A673 cells. Bottom panel, annotations of EWS::FLI1 peaks in heMSCs corresponding to oncogene-bound genes. F Genome browser tracks depicting EWS::FLI1 binding to the PRKCB locus in heMSCs (top) and A673 cells (bottom) . In blue, heMSC-1 cells infected with EWS::FLI1; in gray, heMSC-1 cells infected with control supernatants. Scale, 0-23. Bottom left panel, validation of EWS::FLI1 binding to intron 7 of PRKCB in heMSCs, detected by ChIP-qPCR in EF-heMSC-1 cells. Values referred to the percentage of input and were normalized with respect to the control condition. ACAT1 , negative control. Bottom right panel, PRKCB induction is abolished after EWS::FLI1 knockdown. Data from two independent experiments performed in triplicate are expressed as mean ± s.d. Statistics performed by two-tailed unpaired t -test. G Sankey plots showing annotations of peaks corresponding to genes bound by EWS::FLI1 in both heMSC and A673 cells. In each of the plots, the transitions from distal intergenic (on the left), first intron (in the middle), and other introns (on the right) of the oncogene peaks in heMSCs to the peaks in A673 cells have been highlighted.

Techniques: Infection, ChIP-sequencing, Control, Modification, Binding Assay, Biomarker Discovery, ChIP-qPCR, Negative Control, Knockdown, Two Tailed Test

$A Top panel, genome browser screenshot illustrating EWS::FLI1 binding to the BRCA1 locus in control and EF-heMSC cells. The scale of the tracks is the same size for the control and the EF. Lower panel, chromatin immunoprecipitation of BRCA1 exons 11 and 15 by EWS::FLI1 in heMSC-1 cells infected with EWS::FLI1. Values were referred to the percentage of input and normalized with respect to the control condition. Data correspond to two independent experiments performed in duplicate and are expressed as mean ± s.d. ACAT1, negative control. A two-tailed unpaired t -test was performed. B BRCA1 expression in heMSC-1 cells infected with EWS::FLI1, detected by RT-qPCR and Western blot. Data were obtained from three independent experiments performed in triplicate and expressed as mean ± s.d. A two-tailed unpaired t -test was performed. C BRCA1 induction is abolished after EWS::FLI1 knockdown. Data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. D Representative images of the alkaline comet assay performed with control and EF-heMSC-1 cells. Magnification bar: 50 μm. Below, Box-Whisker plot representation of the quantification of the product of the tail length and the fraction of total DNA in the tail (Olive tail moment) in control and EF-heMSC cells. Box-Whisker plot represents: center line = median; box = 25th–75th percentiles; the lower whisker corresponds to the minimum and the upper whisker to 1,5(75th percentile). Outliers are plotted as individual points. The difference between groups was analyzed by using a multiple regression model and a log( x + 0.1) transformation. E Western blot analysis to detect the expression and phosphorylation status of BRCA1 and kinases involved in DNA damage repair in control and EF-heMSC cells under basal conditions and after treatment with 5 µM etoposide. At the bottom, densitometric quantification of Western blot signals, normalized to Actin intensity ( n = 2 independent experiments). F Dose-response curves and IC50 values for etoposide in control heMSC-1 and EF-heMSC-1 cells. Representative values of three independent experiments are expressed as mean ± s.d. A two-tailed unpaired t -test was performed.$

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Top panel, genome browser screenshot illustrating EWS::FLI1 binding to the BRCA1 locus in control and EF-heMSC cells. The scale of the tracks is the same size for the control and the EF. Lower panel, chromatin immunoprecipitation of BRCA1 exons 11 and 15 by EWS::FLI1 in heMSC-1 cells infected with EWS::FLI1. Values were referred to the percentage of input and normalized with respect to the control condition. Data correspond to two independent experiments performed in duplicate and are expressed as mean ± s.d. ACAT1, negative control. A two-tailed unpaired t -test was performed. B BRCA1 expression in heMSC-1 cells infected with EWS::FLI1, detected by RT-qPCR and Western blot. Data were obtained from three independent experiments performed in triplicate and expressed as mean ± s.d. A two-tailed unpaired t -test was performed. C BRCA1 induction is abolished after EWS::FLI1 knockdown. Data obtained from two independent experiments performed in triplicate are expressed as mean ± s.d. A two-tailed unpaired t -test was performed. D Representative images of the alkaline comet assay performed with control and EF-heMSC-1 cells. Magnification bar: 50 μm. Below, Box-Whisker plot representation of the quantification of the product of the tail length and the fraction of total DNA in the tail (Olive tail moment) in control and EF-heMSC cells. Box-Whisker plot represents: center line = median; box = 25th–75th percentiles; the lower whisker corresponds to the minimum and the upper whisker to 1,5(75th percentile). Outliers are plotted as individual points. The difference between groups was analyzed by using a multiple regression model and a log( x + 0.1) transformation. E Western blot analysis to detect the expression and phosphorylation status of BRCA1 and kinases involved in DNA damage repair in control and EF-heMSC cells under basal conditions and after treatment with 5 µM etoposide. At the bottom, densitometric quantification of Western blot signals, normalized to Actin intensity ( n = 2 independent experiments). F Dose-response curves and IC50 values for etoposide in control heMSC-1 and EF-heMSC-1 cells. Representative values of three independent experiments are expressed as mean ± s.d. A two-tailed unpaired t -test was performed.

Techniques: Binding Assay, Control, Chromatin Immunoprecipitation, Infection, Negative Control, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Alkaline Single Cell Gel Electrophoresis, Whisker Assay, Transformation Assay, Phospho-proteomics

A H&E staining of an abdominal mass and a lung metastasis, and identification of cell clusters based on their corresponding UMAPs. Magnification bars: 400 μm. B Expression maps of some of the most relevant genes that determine clusterization in the above UMAPs. C Expression levels in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ), of the most relevant genes identified by computation of cell clusters from UMAP dimensional reduction. Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed.

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A H&E staining of an abdominal mass and a lung metastasis, and identification of cell clusters based on their corresponding UMAPs. Magnification bars: 400 μm. B Expression maps of some of the most relevant genes that determine clusterization in the above UMAPs. C Expression levels in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ), of the most relevant genes identified by computation of cell clusters from UMAP dimensional reduction. Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed.

Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY

A Expression maps of some of the genes differentially expressed in Ewing sarcomas (Supplementary Data ) (adjusted p value < 0.05, log2(FC), top 100). B IHC of the ES distinctive markers BCL11B and ITM2A in EF-heMSC-derived tumors. Thymus, spleen, and liver sections correspond to normal tissues. Magnification bar, 50 μm. C Expression levels of differentially expressed genes in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ). Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed. D Spatial images of the gene set activity scores calculated using the singscore R package , , which implements a rank-based single-sample scoring method. Scores were computed using unidirectional gene signatures with known direction (knownDirection = TRUE). The resulting scores are directly interpretable as a normalized mean percentile rank. As a reference gene set, the top 400 (for abdominal mass) or 200 (for pulmonary metastasis) differentially expressed genes in Ewing sarcoma (Supplementary Data ) were ordered by the log2(FC) (adjusted p value < 0.5).

Journal: Nature Communications

Article Title: EWS::FLI1 expression in human embryonic mesenchymal stem cells leads to transcriptional reprograming, defective DNA damage repair and Ewing sarcoma

doi: 10.1038/s41467-025-64475-y

Figure Lengend Snippet: A Expression maps of some of the genes differentially expressed in Ewing sarcomas (Supplementary Data ) (adjusted p value < 0.05, log2(FC), top 100). B IHC of the ES distinctive markers BCL11B and ITM2A in EF-heMSC-derived tumors. Thymus, spleen, and liver sections correspond to normal tissues. Magnification bar, 50 μm. C Expression levels of differentially expressed genes in Ewing sarcoma cell lines and in other tumor cell lines, extracted from the DepMap portal ( https://depmap.org/ ). Legends in bold highlight genes directly bound by EWS::FLI1 in A673 cells in promoters or enhancers . A two-tailed Mann–Whitney U test was performed. D Spatial images of the gene set activity scores calculated using the singscore R package , , which implements a rank-based single-sample scoring method. Scores were computed using unidirectional gene signatures with known direction (knownDirection = TRUE). The resulting scores are directly interpretable as a normalized mean percentile rank. As a reference gene set, the top 400 (for abdominal mass) or 200 (for pulmonary metastasis) differentially expressed genes in Ewing sarcoma (Supplementary Data ) were ordered by the log2(FC) (adjusted p value < 0.5).

Techniques: Expressing, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Activity Assay

The set of genes upregulated by AP-1 is enriched for EMT genes and genes repressed by EWS-FLI1. ( A ) EW8 and TC71 cell lines that express doxycycline-inducible c-Jun and c-Fos (TO-Fos/Jun) were grown in the presence or absence of doxycycline for 72 h. Whole-cell lysates were collected for immunoblotting. ( B ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown in the presence or absence of doxycycline for 10–12 days, after which time colonies were stained with crystal violet and counted using an inverted Olympus CKX41 microscope. ** indicates p < 0.001 and **** indicates p < 0.0001. ( C , D ) Volcano plots of differentially expressed genes (Log 2 (Fold Change) > 1 or <−1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun ( C ) and TO-TC71-Fos/Jun ( D ) cell lines cultured in the presence of doxycycline for 72 h hours compared to cell lines cultured in the presence of the vehicle. ( E ) Venn diagram illustrating the overlap between upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells grown in the presence of doxycycline for 72 h compared to cell lines cultured in the presence of the vehicle. ( F , G ) Gene sets (HallmarkMSigDB) enriched in the differentially upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun ( F ) and TO-TC71-Fos/Jun ( G ) cell lines grown in the presence of doxycycline for 72 h compared to vehicle. ( H ) Gene sets (HallmarkMSigDB) enriched in the 1427 genes upregulated in both the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cell lines grown in the presence of doxycycline represented in ( E ). ( I ) Plot demonstrating significant enrichment in ECM-related gene sets among the genes that were significantly upregulated in the TO-EW8-Fos/Jun cells and TO-TC71-Fos/Jun cells grown in the presence of doxycycline for 72 h (adjusted p -value < 0.05). Gene sets were derived from KEGG (Extracellular matrix-receptor interaction, Focal adhesion) and GO: Molecular Function (Extracellular matrix structural constituent) analyses. ( J ) Gene sets (TF.Target.TF.Perturbations.Followed.by.Expression) enriched in the 1427 overlapping upregulated genes represented in ( E ).

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: The set of genes upregulated by AP-1 is enriched for EMT genes and genes repressed by EWS-FLI1. ( A ) EW8 and TC71 cell lines that express doxycycline-inducible c-Jun and c-Fos (TO-Fos/Jun) were grown in the presence or absence of doxycycline for 72 h. Whole-cell lysates were collected for immunoblotting. ( B ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown in the presence or absence of doxycycline for 10–12 days, after which time colonies were stained with crystal violet and counted using an inverted Olympus CKX41 microscope. ** indicates p < 0.001 and **** indicates p < 0.0001. ( C , D ) Volcano plots of differentially expressed genes (Log 2 (Fold Change) > 1 or <−1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun ( C ) and TO-TC71-Fos/Jun ( D ) cell lines cultured in the presence of doxycycline for 72 h hours compared to cell lines cultured in the presence of the vehicle. ( E ) Venn diagram illustrating the overlap between upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells grown in the presence of doxycycline for 72 h compared to cell lines cultured in the presence of the vehicle. ( F , G ) Gene sets (HallmarkMSigDB) enriched in the differentially upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the TO-EW8-Fos/Jun ( F ) and TO-TC71-Fos/Jun ( G ) cell lines grown in the presence of doxycycline for 72 h compared to vehicle. ( H ) Gene sets (HallmarkMSigDB) enriched in the 1427 genes upregulated in both the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cell lines grown in the presence of doxycycline represented in ( E ). ( I ) Plot demonstrating significant enrichment in ECM-related gene sets among the genes that were significantly upregulated in the TO-EW8-Fos/Jun cells and TO-TC71-Fos/Jun cells grown in the presence of doxycycline for 72 h (adjusted p -value < 0.05). Gene sets were derived from KEGG (Extracellular matrix-receptor interaction, Focal adhesion) and GO: Molecular Function (Extracellular matrix structural constituent) analyses. ( J ) Gene sets (TF.Target.TF.Perturbations.Followed.by.Expression) enriched in the 1427 overlapping upregulated genes represented in ( E ).

Article Snippet: Antibodies to the following proteins were used in the immunoblots: c-Jun (60A8, Cell Signaling Technology, #9165, 1:1000; RRID:AB_2130165), FLI1 (Abcam, #ab133485, 1:1000, RRID:AB_2722650); c-Fos (Santa Cruz #sc-271243, 1:100; RRID:AB_10610067), FOSL2/Fra2 (Cell Signaling Technology, #19967, 1:1000; RRID:AB_2722526), Lamin A/C (Developmental Studies Hybridoma Bank, #MANLAC1, 1:100; RRID:AB_2618203), actin (Cell Signaling Technology, #4970, 1:5000; RRID:AB_2223172), vinculin (Proteintech, 26520-1-AP, 1:5000; RRID:AB_2868558), alpha tubulin (Proteintech Cat #66031-1-Ig, 1:5000, RRID:AB_11042766), vimentin (Developmental Studies Hybridoma Bank, #AMF-17b, 1:1000; RRID:AB_528505), FN1 (Cell Signaling Technology, #26836 (also 26836BF), 1:1000, RRID:AB_2924220), Histone H3 (Cell Signaling Technology, #4499 (also 4499S, 4499P, 4499L), 1:1000, RRID:AB_10544537), and FAP (E1V9V, Cell Signaling Technology, #66562, 1:1000, RRID:AB_2904193).

Techniques: Western Blot, Staining, Microscopy, Cell Culture, Derivative Assay, Expressing

Genes repressed by EWS-FLI1 are enriched for EMT-related gene sets. ( A ) EW8, TC71, and A673 cells were treated with siRNA against EWS-FLI1 or a non-targeting control siRNA for 48 h, and then lysates were collected for immunoblotting. ( B ) Log 2 fold change (FC) in EWS-FLI1 mRNA in EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h. The results are representative of three independent experiments. Error bars represent the mean ± SD of three experimental replicates. ( C , D ) Volcano plots of differentially expressed genes (Log 2 (Fold Change) > 1 or <−1, adjusted p -value < 0.05) in the EW8 ( C ) and TC71 ( D ) cell lines treated with siRNA against EWS-FLI1 for 48 h compared to cell lines treated with non-targeting control siRNA. ( E ) Venn diagram demonstrating the overlap between genes that were differentially upregulated (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h compared to genes in the EW8 and TC71 cell lines treated with non-targeting control siRNA. ( F , G ) Gene sets (HallmarkMSigDB) enriched in the differentially upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the EW8 ( C ) and TC71 ( D ) cell lines treated with siRNA against EWS-FLI1 for 48 h compared to cell lines treated with non-targeting control siRNA. ( H ) Gene sets (HallmarkMSigDB) enriched in the 1387 overlap genes that are upregulated in both the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h compared to those in cell lines treated with non-targeting control siRNA represented in ( E ).

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: Genes repressed by EWS-FLI1 are enriched for EMT-related gene sets. ( A ) EW8, TC71, and A673 cells were treated with siRNA against EWS-FLI1 or a non-targeting control siRNA for 48 h, and then lysates were collected for immunoblotting. ( B ) Log 2 fold change (FC) in EWS-FLI1 mRNA in EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h. The results are representative of three independent experiments. Error bars represent the mean ± SD of three experimental replicates. ( C , D ) Volcano plots of differentially expressed genes (Log 2 (Fold Change) > 1 or <−1, adjusted p -value < 0.05) in the EW8 ( C ) and TC71 ( D ) cell lines treated with siRNA against EWS-FLI1 for 48 h compared to cell lines treated with non-targeting control siRNA. ( E ) Venn diagram demonstrating the overlap between genes that were differentially upregulated (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h compared to genes in the EW8 and TC71 cell lines treated with non-targeting control siRNA. ( F , G ) Gene sets (HallmarkMSigDB) enriched in the differentially upregulated genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) in the EW8 ( C ) and TC71 ( D ) cell lines treated with siRNA against EWS-FLI1 for 48 h compared to cell lines treated with non-targeting control siRNA. ( H ) Gene sets (HallmarkMSigDB) enriched in the 1387 overlap genes that are upregulated in both the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h compared to those in cell lines treated with non-targeting control siRNA represented in ( E ).

Techniques: Control, Western Blot

Differential levels of mRNA expression of AP-1 family members in Ewing sarcoma cell lines after siRNA-mediated knockdown of EWS-FLI1 (FDR p -value < 0.05).

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: Differential levels of mRNA expression of AP-1 family members in Ewing sarcoma cell lines after siRNA-mediated knockdown of EWS-FLI1 (FDR p -value < 0.05).

Techniques: Expressing, Knockdown

The list of genes upregulated by RRM1 loss, AP-1 overexpression, and EWS-FLI1 knockdown is enriched for EMT genes. ( A ) Venn diagram demonstrating the 213-gene overlap between differentially expressed genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) that were upregulated in the TO-EW8-Fos/Jun cells grown in doxycycline for 72 h, the EW8 cells treated with siRNA against EWS-FLI1 for 48 h, and the EW8-RRM1-KO cell line grown in the absence of doxycycline for 48 h. ( B ) Venn diagram demonstrating the 72-gene overlap between, differentially expressed genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) that were upregulated in the TO-TC71-Fos/Jun cells grown in doxycycline for 72 h, the TC71 cells treated with siRNA against EWS-FLI1 for 48 h, and the TC71-RRM1-KO cells grown in the absence of doxycycline for 48 h. ( C ) Gene sets (HallmarksMSigDB) enriched in the 213-gene overlap represented in ( A ). ( D ) Gene sets (HallmarksMSigDB) enriched in the 72-gene overlap represented in ( B ). ( E ) Venn diagram demonstrating the 27-gene overlap between ( A , B ). ( F ) Gene sets (HallmarkMSigDB) enriched in the 27 overlapping genes represented in ( E ). ( G – I ) Plots demonstrating significant enrichment of ECM-related gene sets among the genes that were significantly upregulated in the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells grown in doxycycline for 72 h, the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h, and the TC71-RRM1-KO and EW8-RRM1-KO cell lines grown in the absence of doxycycline for 48 h (ShinyGO 0.77, adjusted p -value < 0.05).

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: The list of genes upregulated by RRM1 loss, AP-1 overexpression, and EWS-FLI1 knockdown is enriched for EMT genes. ( A ) Venn diagram demonstrating the 213-gene overlap between differentially expressed genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) that were upregulated in the TO-EW8-Fos/Jun cells grown in doxycycline for 72 h, the EW8 cells treated with siRNA against EWS-FLI1 for 48 h, and the EW8-RRM1-KO cell line grown in the absence of doxycycline for 48 h. ( B ) Venn diagram demonstrating the 72-gene overlap between, differentially expressed genes (Log 2 (Fold Change) > 1, adjusted p -value < 0.05) that were upregulated in the TO-TC71-Fos/Jun cells grown in doxycycline for 72 h, the TC71 cells treated with siRNA against EWS-FLI1 for 48 h, and the TC71-RRM1-KO cells grown in the absence of doxycycline for 48 h. ( C ) Gene sets (HallmarksMSigDB) enriched in the 213-gene overlap represented in ( A ). ( D ) Gene sets (HallmarksMSigDB) enriched in the 72-gene overlap represented in ( B ). ( E ) Venn diagram demonstrating the 27-gene overlap between ( A , B ). ( F ) Gene sets (HallmarkMSigDB) enriched in the 27 overlapping genes represented in ( E ). ( G – I ) Plots demonstrating significant enrichment of ECM-related gene sets among the genes that were significantly upregulated in the TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells grown in doxycycline for 72 h, the EW8 and TC71 cells treated with siRNA against EWS-FLI1 for 48 h, and the TC71-RRM1-KO and EW8-RRM1-KO cell lines grown in the absence of doxycycline for 48 h (ShinyGO 0.77, adjusted p -value < 0.05).

Techniques: Over Expression, Knockdown

Genes repressed by EWS-FLI1 are enriched for ECM components in publicly available EWS-FLI1-knockdown data sets. ( A ) Plot demonstrating significant enrichment in ECM structural components among upregulated genes across three Ewing sarcoma cell lines (A673, EW24, and SK-N-MC) treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE7007, adjusted p -value < 0.05). ( B ) Plot demonstrating significant enrichment in ECM structural components among upregulated genes across four Ewing sarcoma cell lines (A673, MHH-ES1, SKNMC, and TC71) treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE164372, adjusted p -value < 0.05). ( C ) Plot demonstrating significant enrichment in ECM-related gene sets (ECM structural constituent, ECM-receptor interaction, focal adhesion) among significantly upregulated genes in CHLA-10 cells treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE113604, adjusted p -value < 0.05). ( D – F ) Plots demonstrating significant enrichment in ECM-related gene sets among significantly upregulated genes across a panel of Ewing sarcoma cell lines treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE176190, adjusted p -value < 0.05).

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: Genes repressed by EWS-FLI1 are enriched for ECM components in publicly available EWS-FLI1-knockdown data sets. ( A ) Plot demonstrating significant enrichment in ECM structural components among upregulated genes across three Ewing sarcoma cell lines (A673, EW24, and SK-N-MC) treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE7007, adjusted p -value < 0.05). ( B ) Plot demonstrating significant enrichment in ECM structural components among upregulated genes across four Ewing sarcoma cell lines (A673, MHH-ES1, SKNMC, and TC71) treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE164372, adjusted p -value < 0.05). ( C ) Plot demonstrating significant enrichment in ECM-related gene sets (ECM structural constituent, ECM-receptor interaction, focal adhesion) among significantly upregulated genes in CHLA-10 cells treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE113604, adjusted p -value < 0.05). ( D – F ) Plots demonstrating significant enrichment in ECM-related gene sets among significantly upregulated genes across a panel of Ewing sarcoma cell lines treated with shRNA or siRNA against EWS-FLI1 (ShinyGO 0.77, GSE176190, adjusted p -value < 0.05).

Techniques: Knockdown, shRNA

AP-1 overexpression and EWS-FLI1 knockdown upregulate protein expression of ECM components. ( A ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown for 72 h in the presence or absence of doxycycline, and then cellular lysates were collected for immunoblotting. ( B ) EW8, TC71, and A673 cells were treated with siRNA against EWS-FLI1 for 48 h, after which point cellular lysates were collected for immunoblotting. ( C , D ) Plots showing tissue types enriched in TO-EW8-Fos/Jun ( C ) and TO-TC71-Fos/Jun ( D ) cells grown in doxycycline for 72 h compared to vehicle-treated controls (Enrichr, ARCHS 4 Tissues). ( E ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown for 72 h in the presence or absence of doxycycline. Black arrows denote elongated cells with an atypical morphology.

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: AP-1 overexpression and EWS-FLI1 knockdown upregulate protein expression of ECM components. ( A ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown for 72 h in the presence or absence of doxycycline, and then cellular lysates were collected for immunoblotting. ( B ) EW8, TC71, and A673 cells were treated with siRNA against EWS-FLI1 for 48 h, after which point cellular lysates were collected for immunoblotting. ( C , D ) Plots showing tissue types enriched in TO-EW8-Fos/Jun ( C ) and TO-TC71-Fos/Jun ( D ) cells grown in doxycycline for 72 h compared to vehicle-treated controls (Enrichr, ARCHS 4 Tissues). ( E ) TO-EW8-Fos/Jun and TO-TC71-Fos/Jun cells were grown for 72 h in the presence or absence of doxycycline. Black arrows denote elongated cells with an atypical morphology.

Techniques: Over Expression, Knockdown, Expressing, Western Blot

Working model demonstrating the convergence of genes upregulated by EWS-FLI1 knockdown, inhibition or loss of RNR activity, and AP-1 signaling on extracellular-matrix-related signaling pathways in Ewing sarcoma cells. Inhibition or loss of RNR activity in Ewing sarcoma cells results in upregulation of AP-1 protein expression and activity, which subsequently upregulates expression of extracellular-matrix and fibroblast-associated genes, including FN1, vinculin, vimentin, and FAP. These ECM-related genes significantly overlap with EWS-FLI1-repressed genes, demonstrating a form of reciprocal regulation of target genes between AP-1 and EWS-FLI1. Additionally, knockdown of EWS-FLI1 results in the differential expression of various AP-1 proteins at the mRNA and protein levels, suggesting that EWS-FLI1 plays a regulatory role in AP-1 signaling. However, whether this is mechanism is direct or indirect remains to be determined.

Journal: International Journal of Molecular Sciences

Article Title: EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells

doi: 10.3390/ijms25168595

Figure Lengend Snippet: Working model demonstrating the convergence of genes upregulated by EWS-FLI1 knockdown, inhibition or loss of RNR activity, and AP-1 signaling on extracellular-matrix-related signaling pathways in Ewing sarcoma cells. Inhibition or loss of RNR activity in Ewing sarcoma cells results in upregulation of AP-1 protein expression and activity, which subsequently upregulates expression of extracellular-matrix and fibroblast-associated genes, including FN1, vinculin, vimentin, and FAP. These ECM-related genes significantly overlap with EWS-FLI1-repressed genes, demonstrating a form of reciprocal regulation of target genes between AP-1 and EWS-FLI1. Additionally, knockdown of EWS-FLI1 results in the differential expression of various AP-1 proteins at the mRNA and protein levels, suggesting that EWS-FLI1 plays a regulatory role in AP-1 signaling. However, whether this is mechanism is direct or indirect remains to be determined.

Techniques: Knockdown, Inhibition, Activity Assay, Expressing

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