fli1  (Santa Cruz Biotechnology)

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

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.

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

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.

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: 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

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.

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

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).

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, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Activity Assay