Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) EwS TMA sections stained for CD99 (tumor marker) with the low (left) and high (right) pSMAD2 S465/S467 signal. ( B ) Nuclear pSMAD2 S465/467 and cytoplasmic/membranous CD99 IF average intensity per segmented cell. Gates define positive versus negative populations (conservative threshold of pSMAD2 or CD99 true positives). Red box: CD99+ (likely tumor cell) population ( C ) % CD99+ cells that were also pSMAD2+ for each sample in the TMA ( n = 27, table S2); red line: median. ( D ) H&E and IF from EwS TMA sections stained for CD99, pSMAD2 S465/467, and TNC (ECM protein). Right: Zoomed insets showing the CD99 hi /pSMAD low /TNC low region versus CD99 hi /pSMAD2 hi /TNC hi region. ( E ) H&E of the tumor formed 3 weeks post–renal subcapsular injection of A673 cells in NSG mice. Right: IF for pSMAD2 S465/467 and ECM marker TNC (human-specific antibody).

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Staining, Marker, Injection

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) Real-time measurements of confluence (Incucyte) for TC71, A673, CHLA10, and PDX305 cells treated with vehicle controls, TGFβ1 (10 ng/ml), vactosertib (1 μM), or TGFβ1 + vactosertib. n = 2; error bars: SEM. P values: unpaired t tests of confluence at 48 hours relative to 0 hours within each line (nonsignificant comparisons not shown). hr, hours. ( B ) Measurement of the cell area at 48 versus 0 hours of Incucyte imaging in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 2. P values: unpaired t test within each line. ( C ) Cell viability measurements by CellTiter-Glo after 72 hours of culture with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM) in parental cell lines or BlastR control versus dominant-negative TGFBR2 transduced CHLA10 cells. n = 3; P values: unpaired t test within each line. ( D ) Soft agar colony-forming assays of A673 (day 14) and CHLA10 (day 14) cells treated with vehicle control, TGFβ1 (10 ng/ml), or vactosertib (1 μM). ( E ) Number of colonies formed in soft agar for TC71 (day 14), A673 (day 14), CHLA10 (day 14), and PDX305 cells (day 35), relative to the number of colonies in the vehicle control well. n = 2; P values: unpaired t tests within each line. ( F ) Invasive morphology of spheroids cultured for 4 days in 3D rat tail collagen I–rich gels treated with vehicle controls, TGFβ1 (10 ng/ml), or vactosertib (10 μM). Stained with phalloidin to mark F-actin. Yellow arrows: invasive strands. Representative of n = 2. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. hr, hours.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Imaging, Control, Dominant Negative Mutation, Cell Culture, Staining

Journal: Science Advances

Article Title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

doi: 10.1126/sciadv.ady0550

Figure Lengend Snippet: ( A ) % GFP+ after transduction with SBE-GFP SMAD reporter −/+ TGFβ1 (48 hours, n = 2). ( B ) TGFB1 and TGFB2 expression in CAF-like (top 30% expression CAF-like gene signature) versus non-CAF-like cells by scSeq (nine EwS lines). ( C ) ELISAs of TGFβ1 in conditioned media (3 days of culture). n = 3. Dashed line indicates the TGFβ1 level detected in media-only controls. ( D ) ELISAs of secreted total TGFβ2 in conditioned media (3 days of culture). n = 3. TGFβ2 not detected in media-only controls. ( E ) Genes significantly ( P adj < 0.05) up-regulated by TGFβ1 or TGFβ2 in A673, CHLA10, and PDX305 (bulk RNA-seq, n = 3). ( F ) Crystal violet–stained cells 24 hours after seeding on Matrigel-coated transwells to measure invasion. Each cell marked with an asterisk. ( G ) Quantification of Matrigel-coated transwell invasion 24 hours postseeding ( n = 3 to 4; each dot is a biological replicate). P values: unpaired t tests within each line. ( H and I ) ELISAs of secreted total (active and latent) (H) TGFβ2 and (I) TGFβ1 after 3 days of culture of CHLA10 dominant-negative TGFBR2 or empty vector transduced cells. n = 3. P values: unpaired t tests. ( J ) Expression of TNC , TGFB1 , and TGFB2 in PDX305 cells treated with TGFβ1 (10 ng/ml) −/+ vactosertib, TGFβ2 (10 ng/ml) −/+ vactosertib, or vactosertib alone (1 μM). n = 2; P values: unpaired t tests of treatment versus vehicle (nonsignificant comparisons not shown). ( K ) Expression of TGFβ-induced genes in CHLA10 cells treated with TGFβ ligand–blocking antibodies (10 μg/ml, 4 days). n = 2; P values: unpaired t tests. ( L ) IF of TNC in CHLA10 cells treated with TGβ ligand–blocking antibodies versus IgG control (10 μg/ml, 4 days). P values: unpaired t tests. For all panels: n.s., P > 0.05; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: EwS cell lines A673, TC71, and CHLA10 were obtained from American Type Culture Collection (ATCC) and Children's Oncology Group (COG) ( https://childrensoncologygroup.org/ ) cell line repositories.

Techniques: Transduction, Expressing, RNA Sequencing, Staining, Dominant Negative Mutation, Plasmid Preparation, Blocking Assay, Control

Journal: Frontiers in Oncology

Article Title: Construction of a glycosylation-related prognostic signature for predicting prognosis, tumor microenvironment, and immune response in soft tissue sarcoma

doi: 10.3389/fonc.2025.1636830

Figure Lengend Snippet: qPCR validation of 12 GRGS model genes and STT3A regulates the proliferation and viability of STS cells. (A) Relative mRNA expression levels of the 12 genes included in the GRGS signature were measured via qPCR. mRNA expression profiles were assessed in A673 and SW872 STS cell lines, using MSCs as non-tumor controls. (B, C) The expression levels by qPCR of STT3A in STS cells after STT3A knockdown. (D) The expression levels by Western Blotting of STT3A in STS cells after STT3A knockdown. The viability capacity of STS cells after STT3A knockdown was assessed using the CCK-8 assay (E, F) . The colony performed assay (G–I) was conducted to assess the proliferative potential of STS cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: In this experiment, human rhabdomyosarcoma cell line A673 and human liposarcoma cell line SW872 were purchased from Pricella Biotechnology (China), while Human Bone Marrow-derived MSCs (PCS-500-012) were purchased from ATCC (United States) and used as normal controls for comparison with STS cell lines in gene expression.

Techniques: Biomarker Discovery, Expressing, Knockdown, Western Blot, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: Construction of a glycosylation-related prognostic signature for predicting prognosis, tumor microenvironment, and immune response in soft tissue sarcoma

doi: 10.3389/fonc.2025.1636830

Figure Lengend Snippet: STT3A knockdown inhibits the migration and invasion abilities of STS cells. Wound healing assay (A–C) was performed to assess the migration abilities of STS cells after STT3A silencing. Transwell assay (D–H) were conducted to assess the migration and invasion abilities of STS cells by Transwell assay. (D) Representative microscopic images of the Transwell assay. (E, F) Quantification of migration and invasion in A673 cells. (G, H) Quantification of migration and invasion in SW872 cells. Data are presented as mean ± SD. ** P < 0.01; *** P < 0.001.

Article Snippet: In this experiment, human rhabdomyosarcoma cell line A673 and human liposarcoma cell line SW872 were purchased from Pricella Biotechnology (China), while Human Bone Marrow-derived MSCs (PCS-500-012) were purchased from ATCC (United States) and used as normal controls for comparison with STS cell lines in gene expression.

Techniques: Knockdown, Migration, Wound Healing Assay, Transwell Assay

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Tornado plots of FLI1 ChIP-seq in A673 and SKNMC cell lines at 1785 defined EWS::FLI1 enhancer regions. ( B ) Homer Motif analysis showing top 3 enriched motifs in FLI1 ChIP-seq in SKNMC, EWS-502 and A673 cell lines. ( C ) Genomic location of ChIP-seq peaks called using MACS2 of chromatin factors in A673 cells. ( D ) Homer Motif analysis showing top 3 enriched motifs in CBP, p300 and MLL4 ChIP-seq in A673 cells. ( E ) Tornado plots showing histone modification ChIP-seq signal in SKNMC cells at EWS::FLI1 enhancer regions. Signal compared to input. ( F ) Tornado plots for H3K4me3 ChIP-seq and associated input in A673 cells at EWS::FLI1 enhancer regions. ( G ) Western blot analysis of p300 and CBP in steady-state A673, SKNMC and EWS-502 cells.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: ChIP-sequencing, Modification, Western Blot

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Tornado plots of p300, CBP and H3K27ac ChIP-seq in A673 cells at EWS::FLI1 enhancer regions. ( B ) Tornado of MLL3/4 complex members and H3K4me1/2 ChIP-seq in A673 cells at EWS::FLI1 enhancer regions. ( C ) ChIP-seq tracks at NKX2-2 enhancer and CCND1 enhancer. Enhancer regions highlighted in orange. ( D ) MACS2 peak overlaps of p300, CBP, MLL4, and UTX with EWS::FLI1 enhancer regions. ( E ) Correlation of peak intensity of EWS::FLI1 with p300, MLL4, UTX, and WDR5. R2 value for each correlation shown on each graph.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: ChIP-sequencing

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) FLI1 western blot analysis of in A673-dHALO cells following 1 μM VH0032 treatment, EWS-502 dTAG cells following 1 μM dTAG-1 treatment and A673 cells following FLI1 genetic deletion for 7 days. ( B ) FLI1 ChIP-qPCR duplicates from two independent experiments in A673-dHALO cells following 1 μM VH0032 treatment. ( C ) FLI1 ChIP-qPCR in EWS-502 dTAG cells following 1 μM dTAG-1 treatment from two biological replicates. ( D – F ) RT-qPCR in ( D ) A673-dHALO cells following 1 μM VH0032 treatment. ( E ) EWS-502 dTAG system following 1 μM dTAG-1 treatment and ( F ) RT-qPCR in A673 cells following 7 days EWS::FLI1 knockout using two independent gRNAs. Error bars represent standard error of the mean from three biological replicates. ( G ) GSEA plots showing negatively enriched EWS::FLI1 gene expression signatures in A673-dHALO VH0032 treated cells, EWS-502 dTAG-1 treated cells and EWS::FLI1 KO in A673 cells. ( H ) Overlap of downregulated EWS::FLI1-sensitive gene targets between A673-dHALO and EWS-502 dTAG degrader cell lines. ( I ) H3K27ac ChIP-qPCR replicate 2 following 6- and 24 h of EWS::FLI1 degradation in EWS-502 dTAG system (for replicate 1 see Fig. ). ( J ) H3K27ac and H3K4me1 ChIP-qPCR following 7 days EWS::FLI1 genetic deletion from two independent biological replicates. ( K ) Tornado plots showing p300, FLI1 and H3K27ac ChIP-seq signal at EWS::FLI1 enhancers in A673-dHALO and EWS-502 dTAG systems following 24 h degradation. A673-dHALO control plots as in Fig. . ( L ) p300 and CBP ChIP-qPCR after 24 h degradation in both degrader settings from at least two biological replicates. ( M ) MLL4, WDR5, UTX and H3K4me1 ChIP-seq signal at EWS::FLI1 enhancers after 24 h 1 μM VH0032 treatment in A673-dHALO system. A673-dHALO control plots as in Fig. . ( N ) UTX and WDR5 normalized rpk at EWS::FLI1-sensitive enhancers in A673-dHALO system. Mann–Whitney U test, ns P = 0.382, *** P = 0.0005. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( O ) Fold change in H3K27ac and H3K4me1 ChIP-seq signal at EWS::FLI1 enhancers following EWS::FLI1 KO in A673 cells. Red line depicts steady-state levels. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Western Blot, ChIP-qPCR, Quantitative RT-PCR, Knock-Out, Gene Expression, ChIP-sequencing, Control, MANN-WHITNEY

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Overlap between downregulated genes in both A673-dHALO and EWS-502 dTAG system (log2FC <-0.5, FDR < 0.05) with 1785 EWS::FLI1 enhancers. ( B ) Fold change in eRNA signal at all EWS::FLI1 enhancers compared to sensitive EWS::FLI1 enhancers in the A673-dHALO and EWS-502 dTAG system. Kruskal–Wallis test, ** P = 0.0089, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. eRNA signal calculated from three biological replicates. ( C , D ) ChIP-seq and RNA-seq tracks in A673-dHALO cells in DMSO control (-) and VH0032 ( + ) 24 h treatment at NKX2-2 and VRK1 enhancer. ( E ) H3K27ac (green) and H3K4me1 (blue) ChIP-qPCR at NKX2-2 enhancer and gene desert control in both degradation systems. Error bars represent standard error of the mean from at least three biological replicates. Replicate 1 shown for H3K27ac in EWS-502 system. Replicate 2 in Fig. . ( F ) FLI1 ChIP-seq read normalized rpk at EWS::FLI1-sensitive enhancers in control and degrader conditions. Mann–Whitney U test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( G ) Normalized rpk for p300 and H3K27ac ChIP-seq at EWS::FLI1-sensitive enhancers in A673-dHALO system. Mann–Whitney U test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( H ) Normalized rpk for p300 and H3K27ac ChIP-seq at EWS::FLI1-sensitive enhancers in EWS-502 dTAG system. Mann–Whitney U test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( I ) Normalized rpk for MLL4 and H3K4me1 ChIP-seq at EWS::FLI1-sensitive enhancers in A673-dHALO system. Mann–Whitney U test, ns P = 0.997, 0.0738, respectively. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( J ) Normalized rpk for MLL4 and H3K4me1 ChIP-seq at EWS::FLI1-sensitive enhancers in EWS-502 dTAG system (right). Mann–Whitney U test, ns P = 0.275, 0.6791, respectively. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. .

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: ChIP-sequencing, RNA Sequencing, Control, ChIP-qPCR, MANN-WHITNEY

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) p300, CBP, H3K27ac and H3K18ac western blot following nuclear extraction, and histone extraction, of SKNMC cells in DMSO control and 6 h 100 nM dCBP-1 treatment at different concentrations. ( B ) H3K27ac and H3K18ac western blot following histone extraction in 6 h 1 μM A-485 treated SKNMC cells. ( C ) H3K18ac normalized rpk at all EWS::FLI1 enhancers compared to EWS::FLI1-sensitive enhancers in DMSO and dCBP-1 (6 h) condition in SKNMC cells. Mann–Whitney U test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( D – F ) Histone modification ChIP-seq normalized rpk at all EWS::FLI1 enhancers and sensitive enhancers in DMSO control and A-485 (6 h) condition in SKNMC cells. Mann–Whitney U test, ** P = 0.089, *** P = 0.004, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( G ) Tornado plots showing H3K18ac ChIP-seq signal at EWS::FLI1 enhancers in DMSO, A-485 and dCBP-1 conditions in SKNMC cells. ( H ) Example ChIP-seq tracks at EGR2 and CCND1 enhancers for H3K18ac in DMSO, A-485 and dCBP-1 conditions in SKNMC cells. ( I ) H3K4me1 normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in DMSO and A-485 condition in SKNMC cells. Mann–Whitney U test, **** P < 0.0001, * P = 0.0113. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( J ) MLL3, MLL4 and H3K4me1 western blot following nuclear extract and histone extraction in sgGFP control and sgMLL3/4 DKO A673 cells. ( K ) Tornado plots showing H3K4me1, H3K4me2 and H3K27ac ChIP-seq signal at EWS::FLI1 enhancers in sgGFP control and sgMLL3/4 DKO setting in SKNMC cells ( L – N ) H3K4me1, H3K4me2 and H3K27ac normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in sgGFP control and sgMLL3/4 DKO setting. Mann–Whitney U test, * P = 0.0421, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. .

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Western Blot, Extraction, Control, MANN-WHITNEY, ChIP-sequencing, Modification

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) p300 ChIP-qPCR in DMSO control and dCBP-1 conditions in SKNMC cells from two biological replicates. ( B ) p300 and CBP western blot from nuclear extracted SKNMC cells treated with A-485 for 6 h and 24 h. ( C ) H3K18ac ChIP-qPCR in SKNMC and A673 following 6 h dCBP-1 treatment from two biological replicates. ( D , E ) H2BK20ac and H3K27ac ChIP-qPCR at NKX2-2 enhancer in SKNMC and A673 following 6 h dCBP-1 treatment. Graphs shown for two biological replicates. ( F ) H3K27ac ChIP-qPCR at NKX2-2 enhancer following dCBP-1 (6 h) treatment from at least three biological replicates. ( G , H ) H3K18ac and H3K27ac ChIP-qPCR at NKX2-2 and CCND1 enhancer in SKNMC cells following 6 h A-485 treatment from at least two biological replicates. Error bars where present depict standard error of the mean from three biological replicates. ( I ) H3K18ac normalized rpk at EWS::FLI1 enhancers in DMSO and A-485 (6 h) condition in EWS-502 cells. ChIP-seq from one biological replicate. ( J ) H3K27ac normalized rpk at EWS::FLI1 enhancers in DMSO and A-485 (6 h) condition in EWS-502 cells. ChIP-seq from one biological replicate. ( K ) Histone modifications at EWS::FLI1 enhancers in sgGFP control and sgMLL3/4 DKO conditions in A673 cells. ( L ) H3K4me1 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC and A673 cells from two biological replicates. ( M ) H3K4me2 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC cells from two biological replicates. ( N ) H3K27ac ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC and A673 cells from two biological replicates. ( O ) H3K27ac ChIP-seq fold change at all EWS::FLI1 enhancers in MLL3/4 DKO and A-485 (6 h) treatment compared to DMSO control in SKNMC cells. Kruskal–Wallis test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: ChIP-qPCR, Control, Western Blot, ChIP-sequencing

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Western blot analysis in dCBP-1, SAHA and combined SAHA and dCBP-1 treatment. ( B ) FLI1 ChIP-qPCR in DMSO, SAHA and combined SAHA and dCBP-1 treatment. Graphs depict two biological replicates. ( C ) H3K27ac ChIP-seq normalized rpk at EWS::FLI1 enhancers in dCBP-1, SAHA and combined SAHA and dCBP-1 treatment. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. ( D ) RT-qPCR at EWS::FLI1 enhancers in SKNMC cells following 24 h treatment of either 100 nM dCBP-1, 1 μM SAHA or dual treatment from two biological replicates. ( E ) RNA polymerase II ChIP-qPCR at NKX2-2 enhancer in SKNMC cells following 6 h A-485 and 6 h dCBP-1 treatment from two biological replicates. ( F ) Fold change in eRNA RNA-seq reads at EWS::FLI1-sensitive enhancers following MLL3/4 DKO compared to A-485 treatment. Mann–Whitney U test, **** P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. eRNA expression calculated from three biological replicates. ( G ) p300 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in A673 cells. Error bars represent standard error of the mean from at least three biological replicates. ( H ) Western blot analysis of EWS::FLI1 from nuclear extracts of SKNMC cells treated with dCBP-1, A-485 or CCS1477. ‘L’ indicates protein ladder lane. ( I ) EWS::FLI1 ChIP-qPCR at NKX2-2 and CCND1 enhancers in SKNMC cells following 6 h A-485 treatment from two biological replicates.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Western Blot, ChIP-qPCR, ChIP-sequencing, Quantitative RT-PCR, RNA Sequencing, MANN-WHITNEY, Expressing, Control

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) RT-qPCR for mRNA of NKX2-2, CCND1 and PPP1R1A genes following 6 h treatment of A-485, CCS1477 and dCBP-1 treatment in SKNMC cells. Error bars represent standard error of the mean from at least three biological replicates. Kruskal–Wallis test, exact P values indicated on figure. ( B , C ) Volcano plot depicting differential gene expression following 6 h A-485 and dCBP-1 treatment. Red dots and dashed lines represent genes with a log2FC of >−/+1. Gene expression data calculated from three biological replicates. Significant values measured using a negative binomial wald test. ( D ) Gene set enrichment scores showing top 20 negatively enriched gene sets following 6 h A-485 and 6 h dCBP-1 treatment, in order of enrichment in SKNMC cells. Color represents P .adjust values from a Benjamini-Hochberg (BH) procedure test. Gene ratio represents total differentially expressed genes in given gene set. ( E ) Z-scores of A673-sensitive EWS::FLI1 genes in DMSO, dCBP-1 and A-485 treatment conditions. ( F ) Overlap between downregulated A673-dHALO and EWS-502 dTAG-sensitive genes and genes downregulated following A-485 treatment (6 h). ( G ) Overlap between downregulated A673-dHALO and EWS-502 dTAG-sensitive genes and genes downregulated following dCBP-1 treatment (6 h). ( H ) Volcano plot depicting differential gene expression following 7 days of MLL3/4 DKO in A673 cells. Red dots and dashed lines represent genes with a log2FC of <−/+1, FDR < 0.05. Significant values measured using a negative binomial wald test Gene expression data calculated from three biological replicates. ( I ) Overlap between all, and sensitive, downregulated EWS::FLI1-sensitive genes and genes downregulated following MLL3/4 DKO (6 h). .

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Quantitative RT-PCR, Gene Expression

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Overlap between downregulated genes in A-485 and dCBP-1 conditions at two different log2FC cut off thresholds. ( B ) GSEA negatively enriched EWS::FLI1 signatures in both conditions. ( C ) Gene set enrichment scores showing top 20 negatively enriched gene sets following 6 h A-485 and 6 h dCBP-1 treatment, in order of enrichment in EWS-502 cells. Color represents P .adjust values from a Benjamini-Hochberg (BH) procedure test. Gene Ratio represents total differentially expressed genes in given gene set. ( D ) Z-scores of EWS::FLI1-sensitive enhancers and genes in DMSO, dCBP-1 and A-485 treatment conditions. ( E ) RT-qPCR at EWS::FLI1 genes in SKNMC cells following 24 h treatment of either 100 nM dCBP-1, 1 μM SAHA or dual treatment from two biological replicates. ( F ) Overlap between MLL3/4 DKO downregulated genes in SKNMC cells with genes downregulated in A673-dHALO setting and EWS::FLI1-sensitive genes. ( G ) Overlap between MLL3/4 DKO downregulated genes in SKNMC cells with genes downregulated in EWS::FLI1 KO setting in A673 cells. ( H ) Overlap between A-485 downregulated genes (either log2FC < -1 or log2FC < −0.5), MLL3/4 DKO downregulated genes (either log2FC < −1 or log2FC < -0.5) and EWS::FLI1 KO sensitive genes.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Quantitative RT-PCR

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Cell viability of SKNMC and A673 cells treated with A-485, CCS1477 and Dual combination for 6 days measured by CellTiter-Glo. Error bars represent standard deviation of the mean from three biological replicates. ( B ) Schematic showing in vivo experimental method. ( C ) Graph showing percentage initial tumor volume of control mice (blue) and treated (red) for 23 days of A-485 treatment. Each point represents an individual mouse with at least four mice per condition at the end-point. Two-way ANOVA, ** P = 0.0024, 0.0010, *** P = 0.0001, 0.0007, **** P < 0.0001. ( D ) Spaghetti plot showing percentage initial tumor volume of individual mice in control (blue) and treated (red) conditions. ( E ) Western blot analysis of H3K27ac and H3K18ac following histone extraction of tumor cells extracted from a vehicle-only and a A-485-treated mouse. ( F ) RT-qPCR at NKX2-2 and CCND1 eRNA and mRNA in vehicle (blue) and treated (red) mice. Each individual point represents expression from a single animal. Error bars represent standard deviation of the mean from at least four biological replicates. Mann–Whitney U test, * P = 0.00242, 0.029, ** P = 0.0073, 0.0012. .

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques: Standard Deviation, In Vivo, Control, Western Blot, Extraction, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Synergy plots for combination treatment of A-485 and CCS1477 in A673, SKNMC and EWS-502 cells. ( B ) Percentage weight of mice compared to day 1 of treatment in vehicle-only (blue) and 100 mg/kg A-485 (red) cohorts. Each point and line represent a single mouse.

Article Snippet: Ewing sarcoma cell lines A673 (ATCC, CRL-1598) and SKNMC (ATCC, HTB-10) cells were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% l -Glutamine (Gibco) and 1× penicillin/streptomycin (Gibco).

Techniques:

Journal: bioRxiv

Article Title: Global Profiling of Remodeled Subcellular Structures Due to Drug Treatment and Disease

doi: 10.1101/2025.08.27.672480

Figure Lengend Snippet: ( A ) DNA poor and low DAPI stain structures in the nucleus is a hallmark of nucleoli and was quantified for a non-Ewing cell line, HEK293, with and without EWS-FLI1 expressed, an Ewing sarcoma cell line, A673, and a non-Ewing osteosarcoma, U2OS. ( B ) Area of DNA poor regions was quantified for nucleoli, and no significant difference was detected among the four cell lines and conditions. ( C ) The number of DNA poor regions in A673 and HEK293 expressing EWS-FLI1 was significantly lower than cells with EWS-FLI1 protein expressed.

Article Snippet: Cell lines A673 (ATCC, CRL-1598), SK-N-MC (ATCC, HTB-10), U2OS (ATCC, HTB-96), and HEK293T/17 (ATCC, CRL-11268) were obtained from ATCC and cultured at 5% CO 2 and 37 oC.

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: Ewing sarcoma cells are sensitive to HSV-TK/GCV-mediated toxicity. (a) A673 and MHH-ES1 Ewing sarcoma cells were infected at an MOI of 0.5 with lentivirus harboring the HSV-TK(SR39h)/HA gene under the control of the constitutive promoter EF1A (EF1A > TK) or the Ewing-specific promoter GGAAprom (GGAA > TK). Western blot confirming the expression of HSV-TK. (b) A673 and MHH-ES1 cells expressing HSV-TK under the aforementioned promoters were incubated with different concentrations of GCV for six days, and the effect on cell viability was quantified with a resazurin assay (mean ± SD of three independent experiments).

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Control, Western Blot, Expressing, Incubation, Resazurin Assay

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: HSV-TK/GCV-mediated bystander effect in Ewing sarcoma cells. (a) A673 and MHH-ES1 cells stably transduced with GGAA > HSV-TK(SR39h) lentivirus were cocultured with cells stably transduced with the luciferase gene (LUC cells). The ratios of GGAA > TK cells to LUC cells are indicated. The cells were incubated with 10 µM GCV, and luciferase activity was quantified after 72 h. Each point represents the mean of one experiment performed in triplicate (3–4 independent experiments). (mean ± SD). (b) MHH-ES1 cells constitutively expressing EGFP and HSV-TK(SR39h) under the control of the GGAA promoter were cocultured with cells constitutively expressing mCherry (50:50 ratio) for 93 h in the absence (control) or presence of GCV (10 µM). Representative fluorescence images showing that HSV-TK-negative cells (mCherry-positive cells) were killed in the presence of HSV-TK-positive cells (EGFP-positive cells). A representative video is also included in the supplementary material.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Stable Transfection, Transduction, Luciferase, Incubation, Activity Assay, Expressing, Control, Fluorescence

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: HSV-TK/GCV therapy reduces tumor growth and induces immune cell infiltration in vivo. (a) Schematic representation of the experimental design. (b) Nude mice were inoculated s.c. with A673/GGAA > HSV-TK (SR39h) cells and split into two groups when the tumors reached a mean volume of approximately 200 mm 3 (dashed line). One group was treated with valganciclovir (VGCV) ( n = 8) dissolved in drinking water, and the other group was treated with vehicle ( n = 6). The tumor volume (mean ± s.e.m) and tumor volume normalized to day 14 (start of treatment) for each animal are shown. (c) Micrograph of tumors excised at the end of the experiment (day 22). Two animals in the treated group presented no residual tumors and thus were missing in the image. (d) Tumor sections were stained with an anti-mouse CD45 antibody to assess immune cell infiltration. Representative images of tumors from the VGCV and control groups demonstrating the massive death of Ewing sarcoma cells and immune cell infiltration (CD45 + cells) in the VGCV group. Images from the control and VGCV groups are shown at the same magnification.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Staining, Control

Journal: Scientific Reports

Article Title: Suicide gene therapy targeting ewing sarcoma via an ewing-specific GGAA promoter

doi: 10.1038/s41598-025-14945-6

Figure Lengend Snippet: The combination of adenovirus GGAA > TK (Ad-GGAA > TK) and VGCV reduces tumor growth and induces immune cell infiltration in vivo. (a) Schematic representation of the experimental design. Nude mice were inoculated s.c. with A673 Ewing sarcoma cells and split into three groups when the tumors reached a mean volume of approximately 200 mm 3 (dashed line). One group received intratumoral injections of PBS (control PBS) ( n = 7), other group received intratumoral injections of control adenovirus (Ad-GGAA > ORF, 2 × 10 10 VP/mL dose) ( n = 6) and the third group received intratumoral administration of the therapeutic adenovirus Ad-GGAA > TK (2 × 10 10 VP/mL dose) ( n = 7). PBS and adenoviruses were administered at 2–3-day intervals (four doses). All groups received VGCV dissolved in drinking water. (b) The tumor volume normalized with respect to the start of treatment for each group and each animal is shown (mean ± s.d.). Statistical significance ( p-values ) between groups is shown (2-way ANOVA, ns = not significant). (c) Micrograph of tumors excised at the end of the experiment for each experimental group. (d) Tumor sections were stained with an anti-mouse CD45 antibody to assess immune infiltration. Representative images of a tumor treated with Ad-GGAA > TK and tumors treated with PBS or the adenovirus control Ad-GGAA > ORF are shown. Control tumors (PBS and Ad-GGAA > ORF) were negative for CD45 + cells except at the periphery of the tumor, where some CD45 + cells accumulated. In contrast, Ad-GGAA > TK treatment induced massive death of Ewing sarcoma cells and immune cell infiltration (CD45 + cells). Images are shown at the same magnification.

Article Snippet: The Ewing sarcoma cell line A673 (CRL-1598), the fibrosarcoma cell line HT1080 (CCL-121) and the osteosarcoma cell lines U2-OS (HTB-96) and Saos-2 (HTB-85) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Control, Staining