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ews cell lines tc71  (DSMZ)


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    DSMZ ews cell lines tc71
    Inhibition of DDX3 radiosensitizes <t>EWS</t> (A) Immunofluorescent images of <t>TC71</t> EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.
    Ews Cell Lines Tc71, supplied by DSMZ, used in various techniques. Bioz Stars score: 95/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ews+cell+lines+tc71/pmc10844834-389-1-44?v=DSMZ
    Average 95 stars, based on 79 article reviews
    ews cell lines tc71 - by Bioz Stars, 2026-08
    95/100 stars

    Images

    1) Product Images from "RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma"

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    Journal: iScience

    doi: 10.1016/j.isci.2024.108925

    Inhibition of DDX3 radiosensitizes EWS (A) Immunofluorescent images of TC71 EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.
    Figure Legend Snippet: Inhibition of DDX3 radiosensitizes EWS (A) Immunofluorescent images of TC71 EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.

    Techniques Used: Inhibition, Staining, Quantitation Assay, Genetically Modified, Irradiation

    DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also <xref ref-type=Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. " title="... SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm.

    Techniques Used: Western Blot, Knockdown, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Homologous Recombination, Non-Homologous End Joining, Immunoprecipitation, Magnetic Beads, Control, Staining

    DDX3 interacts with and modulates cytoplasmic oligonucleotide substrates in EWS (A–C) DAPI staining of dsDNA in three independent EWS cell lines (A), three independent xenografts (B), and three independent patient samples (C). White arrows show examples of extra-nuclear dsDNA substrates. Mag bars: 10 μm. (D) Immunofluorescent images of TC71 EWS cells. Data are representative of two independent experiments. Green = single-stranded DNA (ssDNA); Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (E) Representative images of S9.6 staining of umbilical-cord-derived human mesenchymal stem cells (MSC), TC71, and A4573 EWS cell lines. Mag bar: 10 μm. (F and G) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained, and immunofluorescent (IF) confocal images were obtained (F) and basal RNA:DNA hybrid abundance quantified (G). Results are representative of four to five high power field (hpf) per condition from two independent experiments per cell line. Data represent mean fluorescent intensity per cell per hpf +/− SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by unpaired t tests. Red = HA-tag; Green = RNA:DNA hybrids (S9.6 staining); Blue = DAPI stain. Mag bar: 10 μm. (H) TC71 and A4573 EWS cell lines were transduced with either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained with J2 to visualize dsRNA. Representative IF confocal images are shown. Mag bar: 10 μm. (I and J) Representative IF images of TC71 cells at 1 and 3 h post-2 Gy IR (I). Colocalization of DDX3 and RNA:DNA hybrid fluorescence was analyzed and quantified using Pearson’s coefficient. Data are representative of five high power fields (hpf) per cohort from one of three independent experiments (J). Data are mean ± SEM. ∗∗∗∗p < 0.0001 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Green = DDX3; Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (K and L) TC71 and A4573 cells were treated with either vehicle control (DMSO) or 2 μM RK-33. Immunofluorescent z stack confocal images were obtained (K). TC71 images were measured for volume of RNA:DNA hybrid structures per cell (L). Results are representative of five hpf per condition from two independent experiments. Data represent mean fluorescent intensity per cell +/− SEM. ∗∗∗p < 0.001 determined by unpaired t test. Mag bar: 10 μm. (M) Representative double-stained IF images analyzing RNA:DNA hybrid and double-stranded RNA (dsRNA) distribution with a rabbit-S9.6 and J2 antibodies, respectively, in TC71 and A4573 EWS cell lines treated with either DMSO or 2 μM RK-33. Mag bar: 10 μm.
    Figure Legend Snippet: DDX3 interacts with and modulates cytoplasmic oligonucleotide substrates in EWS (A–C) DAPI staining of dsDNA in three independent EWS cell lines (A), three independent xenografts (B), and three independent patient samples (C). White arrows show examples of extra-nuclear dsDNA substrates. Mag bars: 10 μm. (D) Immunofluorescent images of TC71 EWS cells. Data are representative of two independent experiments. Green = single-stranded DNA (ssDNA); Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (E) Representative images of S9.6 staining of umbilical-cord-derived human mesenchymal stem cells (MSC), TC71, and A4573 EWS cell lines. Mag bar: 10 μm. (F and G) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained, and immunofluorescent (IF) confocal images were obtained (F) and basal RNA:DNA hybrid abundance quantified (G). Results are representative of four to five high power field (hpf) per condition from two independent experiments per cell line. Data represent mean fluorescent intensity per cell per hpf +/− SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by unpaired t tests. Red = HA-tag; Green = RNA:DNA hybrids (S9.6 staining); Blue = DAPI stain. Mag bar: 10 μm. (H) TC71 and A4573 EWS cell lines were transduced with either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained with J2 to visualize dsRNA. Representative IF confocal images are shown. Mag bar: 10 μm. (I and J) Representative IF images of TC71 cells at 1 and 3 h post-2 Gy IR (I). Colocalization of DDX3 and RNA:DNA hybrid fluorescence was analyzed and quantified using Pearson’s coefficient. Data are representative of five high power fields (hpf) per cohort from one of three independent experiments (J). Data are mean ± SEM. ∗∗∗∗p < 0.0001 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Green = DDX3; Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (K and L) TC71 and A4573 cells were treated with either vehicle control (DMSO) or 2 μM RK-33. Immunofluorescent z stack confocal images were obtained (K). TC71 images were measured for volume of RNA:DNA hybrid structures per cell (L). Results are representative of five hpf per condition from two independent experiments. Data represent mean fluorescent intensity per cell +/− SEM. ∗∗∗p < 0.001 determined by unpaired t test. Mag bar: 10 μm. (M) Representative double-stained IF images analyzing RNA:DNA hybrid and double-stranded RNA (dsRNA) distribution with a rabbit-S9.6 and J2 antibodies, respectively, in TC71 and A4573 EWS cell lines treated with either DMSO or 2 μM RK-33. Mag bar: 10 μm.

    Techniques Used: Staining, Derivative Assay, Transduction, Fluorescence, Control

    Inhibition of DDX3 helicase activity sequesters RAD51 in the cytoplasm in an RNA:DNA-hybrid-dependent manner following IR (A) Representative immunofluorescent images of two EWS cell lines, TC71 and A4573, demonstrating cytoplasmic colocalization of endogenous RAD51 (green) and DDX3 (red) at basal levels. Data are representative of three independent experiments. Blue = DAPI stain. Mag bar: 10 μm. (B) Representative immunofluorescent images of TC71 cells demonstrating colocalization of RAD51 (green) with DSBs, evidenced by γ-H2A.X (purple) staining, 3 h following 2 Gy IR. Data are representative of two independent experiments. Red = DDX3; Blue = DAPI stain. Mag bar: 10 μm. (C) Immunofluorescent images of TC71 cells at 3 h following treatment with either DMSO (top), 2 μm RK-33 (second row), DMSO +2 Gy IR (third row), or 2 μM RK-33 + 2 Gy (bottom). Data are representative of three independent experiments. Green = DDX3; Red = RAD51; Cyan = RNA:DNA hybrids; Blue = DAPI stain. Mag bar: 20 μm. (D) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h prior to performing radiosensitization assays. Cells were stained and immunofluorescent, z-stacked confocal images were obtained. RAD51 foci were quantified for each experimental cohort. Results represent nuclear RAD51 foci as a percentage of total RAD51 foci from all Z-planes of 4–5 representative hpf from each experimental cohort of each cell line. Data represent one of three independent experiments. Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-way ANOVA followed by Šídák’s multiple comparisons test.
    Figure Legend Snippet: Inhibition of DDX3 helicase activity sequesters RAD51 in the cytoplasm in an RNA:DNA-hybrid-dependent manner following IR (A) Representative immunofluorescent images of two EWS cell lines, TC71 and A4573, demonstrating cytoplasmic colocalization of endogenous RAD51 (green) and DDX3 (red) at basal levels. Data are representative of three independent experiments. Blue = DAPI stain. Mag bar: 10 μm. (B) Representative immunofluorescent images of TC71 cells demonstrating colocalization of RAD51 (green) with DSBs, evidenced by γ-H2A.X (purple) staining, 3 h following 2 Gy IR. Data are representative of two independent experiments. Red = DDX3; Blue = DAPI stain. Mag bar: 10 μm. (C) Immunofluorescent images of TC71 cells at 3 h following treatment with either DMSO (top), 2 μm RK-33 (second row), DMSO +2 Gy IR (third row), or 2 μM RK-33 + 2 Gy (bottom). Data are representative of three independent experiments. Green = DDX3; Red = RAD51; Cyan = RNA:DNA hybrids; Blue = DAPI stain. Mag bar: 20 μm. (D) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h prior to performing radiosensitization assays. Cells were stained and immunofluorescent, z-stacked confocal images were obtained. RAD51 foci were quantified for each experimental cohort. Results represent nuclear RAD51 foci as a percentage of total RAD51 foci from all Z-planes of 4–5 representative hpf from each experimental cohort of each cell line. Data represent one of three independent experiments. Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-way ANOVA followed by Šídák’s multiple comparisons test.

    Techniques Used: Inhibition, Activity Assay, Staining, Transduction


    Figure Legend Snippet:

    Techniques Used: Control, Plasmid Preparation, Microarray, Recombinant, Transfection, Magnetic Beads, DC Protein Assay, Extraction, Polymer, Software



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    Johns Hopkins HealthCare ewing sarcoma cell line tc71
    Purification and characterization of EV preparations from EwS and fibroblast cell lines and healthy donor plasma. ( A ) Size distribution of EVs isolated by ultrafiltration and differential centrifugation from healthy donor plasma, MRC5 fibroblasts, and A4573, A673, TC32, and <t>TC71</t> EwS cell lines. Nanoparticle tracking analysis of three independent EV preparations (dotted line) and respective mean (solid line) is shown. ( B ) Immunoblotting-detection of chaperon proteins Hsp70 and Hsp90, EV markers CD63, CD81 and TSG101, tubulin and actin in the purified EV preparations from EwS cell lines and healthy donor plasma ( n = 3 independent donors). ( C ) Expression of the CD63 and CD81 EV markers (solid line) compared to IT antibodies (dotted line) on MRC5 EVs or EwS EVs bound to 3.9 µm latex beads. Representative flow cytometry result (a total of 3 independent EV preparations) is shown. ( D ) TNF release from CD14 + monocytes treated for 6 h with three independent EV preparations (3 × 10 9 EVs/mL) from healthy donor plasma, MRC5, or EwS cell lines. Conditioned medium was analyzed by cytokine singleplex assay.
    Ewing Sarcoma Cell Line Tc71, supplied by Johns Hopkins HealthCare, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ewing’s sarcoma cell line tc71  (Lonza)
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    Lonza ewing’s sarcoma cell line tc71
    Antitumor activity of SST0001 against TC71 <t> Ewing’s </t> sarcoma
    Ewing’s Sarcoma Cell Line Tc71, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibition of DDX3 radiosensitizes EWS (A) Immunofluorescent images of TC71 EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.

    Journal: iScience

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    doi: 10.1016/j.isci.2024.108925

    Figure Lengend Snippet: Inhibition of DDX3 radiosensitizes EWS (A) Immunofluorescent images of TC71 EWS cells at 1 or 24 h following 2 Gy IR that were treated with either DMSO (top) or 2 μM RK-33 (bottom). Green = γ-H2A.X detection, marking double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. (B) Quantitation of γ-H2A.X foci in stable genetically modified shDDX3 MHH-ES-1 cell lines 2D7 and 2C7 at 0 (i.e., no treatment), 6, and 24 h following 2 Gy IR. Data are representative of three independent experiments. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (C) Quantitation of γ-H2A.X foci in three independent EWS cell lines (TC71, MHH-ES-1, and TC32) where cells were irradiated with 2 Gy in the presence of 0, 0.5, 1, 2, or 4 μM RK-33. Data represent three independent experiments per cell line. Data are mean ± SD. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-Way ANOVA followed by Šídák’s multiple comparisons test. (D and E) TC71, MHH-ES-1, and A4573 EWS cells were treated with either DMSO, 2 Gy, 2 μM RK-33, or 2 μM RK-33 + 2 Gy and plated at densities of 200 or 400 cells/well 6 h post-IR. Cells were then grown in conditioned media for five days and stained with crystal violet for (D) visualization and (E) quantification of clonogenic survival fractions (n = 6 technical replicates per cohort per cell line). Results represent one experiment of three independent experiments per cell line. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by one-way ANOVA followed by Šídák’s multiple comparisons test.

    Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

    Techniques: Inhibition, Staining, Quantitation Assay, Genetically Modified, Irradiation

    DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also <xref ref-type=Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm. " width="100%" height="100%">

    Journal: iScience

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    doi: 10.1016/j.isci.2024.108925

    Figure Lengend Snippet: DDX3 interacts with components of the homologous DDR pathway (A) Western blots of DR-U2OS and EJ5-U2OS cell lysates following siRNA knockdown of DDX3. (B) DR-U2OS, EJ5-U2OS, and SSA-U2OS cell lines were treated with either scramble or siDDX3 and then transfected with the I Sce I-pCAGGS vector or an empty vector to induce DNA damage. Effective DDR was visualized by induction of GFP expression and quantified using flow cytometry. Results represent three independent experiments per cell line. Data represent frequency of DNA recombination events ±SEM. ∗∗∗p < 0.001 determined by multiple unpaired t tests followed by Šídák’s multiple comparisons test. I, induction of DNA damage with I Sce I-pCAGGS; UI, non-induction with empty pCAGGS vector; HR, homologous recombination; NHEJ, non-homologous end-joining; SSA, single-strand annealing. (C) Immunoprecipitation (IP) of TC71 EWS cell lysates using anti-DDX3 antibodies conjugated to magnetic beads. Western blots demonstrate co-immunoprecipitation of various DDR proteins with endogenous DDX3. Iso IgG, immunoprecipitation of TC71 cell lysates using control isotype antibodies. Results are representative of three independent experiments. See also Figure S2 . (D) Subcellular fractionations of TC71 cells demonstrate the presence of DDX3, RAD51, RECQL1, RPA32, and XRCC2 in both cytoplasmic and nuclear compartments. Results are representative of three independent experiments. See also Figure S2 . CL, whole-cell lysate. (E) Immunofluorescent images of TC71 EWS cells 6 h after treatment with 5 μM RK-33 and 2 Gy IR. Green = γ-H2A.X staining of double-stranded DNA breaks (DSB); Red = DDX3; Blue = DAPI stain. Mag bar: 20 μm.

    Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

    Techniques: Western Blot, Knockdown, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Homologous Recombination, Non-Homologous End Joining, Immunoprecipitation, Magnetic Beads, Control, Staining

    DDX3 interacts with and modulates cytoplasmic oligonucleotide substrates in EWS (A–C) DAPI staining of dsDNA in three independent EWS cell lines (A), three independent xenografts (B), and three independent patient samples (C). White arrows show examples of extra-nuclear dsDNA substrates. Mag bars: 10 μm. (D) Immunofluorescent images of TC71 EWS cells. Data are representative of two independent experiments. Green = single-stranded DNA (ssDNA); Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (E) Representative images of S9.6 staining of umbilical-cord-derived human mesenchymal stem cells (MSC), TC71, and A4573 EWS cell lines. Mag bar: 10 μm. (F and G) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained, and immunofluorescent (IF) confocal images were obtained (F) and basal RNA:DNA hybrid abundance quantified (G). Results are representative of four to five high power field (hpf) per condition from two independent experiments per cell line. Data represent mean fluorescent intensity per cell per hpf +/− SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by unpaired t tests. Red = HA-tag; Green = RNA:DNA hybrids (S9.6 staining); Blue = DAPI stain. Mag bar: 10 μm. (H) TC71 and A4573 EWS cell lines were transduced with either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained with J2 to visualize dsRNA. Representative IF confocal images are shown. Mag bar: 10 μm. (I and J) Representative IF images of TC71 cells at 1 and 3 h post-2 Gy IR (I). Colocalization of DDX3 and RNA:DNA hybrid fluorescence was analyzed and quantified using Pearson’s coefficient. Data are representative of five high power fields (hpf) per cohort from one of three independent experiments (J). Data are mean ± SEM. ∗∗∗∗p < 0.0001 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Green = DDX3; Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (K and L) TC71 and A4573 cells were treated with either vehicle control (DMSO) or 2 μM RK-33. Immunofluorescent z stack confocal images were obtained (K). TC71 images were measured for volume of RNA:DNA hybrid structures per cell (L). Results are representative of five hpf per condition from two independent experiments. Data represent mean fluorescent intensity per cell +/− SEM. ∗∗∗p < 0.001 determined by unpaired t test. Mag bar: 10 μm. (M) Representative double-stained IF images analyzing RNA:DNA hybrid and double-stranded RNA (dsRNA) distribution with a rabbit-S9.6 and J2 antibodies, respectively, in TC71 and A4573 EWS cell lines treated with either DMSO or 2 μM RK-33. Mag bar: 10 μm.

    Journal: iScience

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    doi: 10.1016/j.isci.2024.108925

    Figure Lengend Snippet: DDX3 interacts with and modulates cytoplasmic oligonucleotide substrates in EWS (A–C) DAPI staining of dsDNA in three independent EWS cell lines (A), three independent xenografts (B), and three independent patient samples (C). White arrows show examples of extra-nuclear dsDNA substrates. Mag bars: 10 μm. (D) Immunofluorescent images of TC71 EWS cells. Data are representative of two independent experiments. Green = single-stranded DNA (ssDNA); Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (E) Representative images of S9.6 staining of umbilical-cord-derived human mesenchymal stem cells (MSC), TC71, and A4573 EWS cell lines. Mag bar: 10 μm. (F and G) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained, and immunofluorescent (IF) confocal images were obtained (F) and basal RNA:DNA hybrid abundance quantified (G). Results are representative of four to five high power field (hpf) per condition from two independent experiments per cell line. Data represent mean fluorescent intensity per cell per hpf +/− SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 determined by unpaired t tests. Red = HA-tag; Green = RNA:DNA hybrids (S9.6 staining); Blue = DAPI stain. Mag bar: 10 μm. (H) TC71 and A4573 EWS cell lines were transduced with either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h. Cells were then stained with J2 to visualize dsRNA. Representative IF confocal images are shown. Mag bar: 10 μm. (I and J) Representative IF images of TC71 cells at 1 and 3 h post-2 Gy IR (I). Colocalization of DDX3 and RNA:DNA hybrid fluorescence was analyzed and quantified using Pearson’s coefficient. Data are representative of five high power fields (hpf) per cohort from one of three independent experiments (J). Data are mean ± SEM. ∗∗∗∗p < 0.0001 determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. Green = DDX3; Red = RNA:DNA hybrid structures; Blue = DAPI stain. Mag bar: 24 μm. (K and L) TC71 and A4573 cells were treated with either vehicle control (DMSO) or 2 μM RK-33. Immunofluorescent z stack confocal images were obtained (K). TC71 images were measured for volume of RNA:DNA hybrid structures per cell (L). Results are representative of five hpf per condition from two independent experiments. Data represent mean fluorescent intensity per cell +/− SEM. ∗∗∗p < 0.001 determined by unpaired t test. Mag bar: 10 μm. (M) Representative double-stained IF images analyzing RNA:DNA hybrid and double-stranded RNA (dsRNA) distribution with a rabbit-S9.6 and J2 antibodies, respectively, in TC71 and A4573 EWS cell lines treated with either DMSO or 2 μM RK-33. Mag bar: 10 μm.

    Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

    Techniques: Staining, Derivative Assay, Transduction, Fluorescence, Control

    Inhibition of DDX3 helicase activity sequesters RAD51 in the cytoplasm in an RNA:DNA-hybrid-dependent manner following IR (A) Representative immunofluorescent images of two EWS cell lines, TC71 and A4573, demonstrating cytoplasmic colocalization of endogenous RAD51 (green) and DDX3 (red) at basal levels. Data are representative of three independent experiments. Blue = DAPI stain. Mag bar: 10 μm. (B) Representative immunofluorescent images of TC71 cells demonstrating colocalization of RAD51 (green) with DSBs, evidenced by γ-H2A.X (purple) staining, 3 h following 2 Gy IR. Data are representative of two independent experiments. Red = DDX3; Blue = DAPI stain. Mag bar: 10 μm. (C) Immunofluorescent images of TC71 cells at 3 h following treatment with either DMSO (top), 2 μm RK-33 (second row), DMSO +2 Gy IR (third row), or 2 μM RK-33 + 2 Gy (bottom). Data are representative of three independent experiments. Green = DDX3; Red = RAD51; Cyan = RNA:DNA hybrids; Blue = DAPI stain. Mag bar: 20 μm. (D) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h prior to performing radiosensitization assays. Cells were stained and immunofluorescent, z-stacked confocal images were obtained. RAD51 foci were quantified for each experimental cohort. Results represent nuclear RAD51 foci as a percentage of total RAD51 foci from all Z-planes of 4–5 representative hpf from each experimental cohort of each cell line. Data represent one of three independent experiments. Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-way ANOVA followed by Šídák’s multiple comparisons test.

    Journal: iScience

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    doi: 10.1016/j.isci.2024.108925

    Figure Lengend Snippet: Inhibition of DDX3 helicase activity sequesters RAD51 in the cytoplasm in an RNA:DNA-hybrid-dependent manner following IR (A) Representative immunofluorescent images of two EWS cell lines, TC71 and A4573, demonstrating cytoplasmic colocalization of endogenous RAD51 (green) and DDX3 (red) at basal levels. Data are representative of three independent experiments. Blue = DAPI stain. Mag bar: 10 μm. (B) Representative immunofluorescent images of TC71 cells demonstrating colocalization of RAD51 (green) with DSBs, evidenced by γ-H2A.X (purple) staining, 3 h following 2 Gy IR. Data are representative of two independent experiments. Red = DDX3; Blue = DAPI stain. Mag bar: 10 μm. (C) Immunofluorescent images of TC71 cells at 3 h following treatment with either DMSO (top), 2 μm RK-33 (second row), DMSO +2 Gy IR (third row), or 2 μM RK-33 + 2 Gy (bottom). Data are representative of three independent experiments. Green = DDX3; Red = RAD51; Cyan = RNA:DNA hybrids; Blue = DAPI stain. Mag bar: 20 μm. (D) Three independent EWS cell lines, TC71, A4573, and MHH-ES-1, were transduced with lentivirus overexpressing either RNaseH1 WT or enzymatically dead RNaseH1 D210N for 48 h prior to performing radiosensitization assays. Cells were stained and immunofluorescent, z-stacked confocal images were obtained. RAD51 foci were quantified for each experimental cohort. Results represent nuclear RAD51 foci as a percentage of total RAD51 foci from all Z-planes of 4–5 representative hpf from each experimental cohort of each cell line. Data represent one of three independent experiments. Data are mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 determined by two-way ANOVA followed by Šídák’s multiple comparisons test.

    Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

    Techniques: Inhibition, Activity Assay, Staining, Transduction

    Journal: iScience

    Article Title: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma

    doi: 10.1016/j.isci.2024.108925

    Figure Lengend Snippet:

    Article Snippet: Established EWS cell lines TC71 (RRID:CVCL_2213) and TC32 (RRID:CVCL_7151) were acquired from Children’s Cancer Respository ( https://cccells.org ), A4573 (RRID:CVCL_6245) was a kind gift from the laboratory of Katia Scotlandi, and MHH-ES-1 (ACC-167; RRID:CVCL_1411) was purchased from German Collection of Microorganisms and Cell Cultures (dsmz.de, Braunschweig, Germany).

    Techniques: Control, Plasmid Preparation, Microarray, Recombinant, Transfection, Magnetic Beads, DC Protein Assay, Extraction, Polymer, Software

    Figure 1. STAG2 knockout profoundly alters the transcriptomic landscape (A) Representative western blotting in cellular extracts from isogenic STAG2 knockout (KO) (generated with two independent sgRNAs: SA2m#1 and SA2m#2), STAG1 KO (sgRNA: SA1m#1), STAG2 rescued (sgRNA: SA2r) and STAG2 knockdown (KD) at 48 h (generated with two independent siRNAs: siSA2#6 and siSA2#8) Ewing sarcoma cells. Color code for sgRNA isogenic models is indicated for each model and kept identical throughout the article. (B) Scaled Venn diagram for modulated genes between STAG2 WT and KO conditions (n = 3); total modulated genes for each condition represent the sum of intra- circle numbers; universe includes expressed genes (n = 13,780). p value for intersection was calculated with the SuperExact test. (C) Boxplots of log2 fold change for up-, un-, and downregulated genes in STAG2 KO and STAG2 rescued cells as compared with A673 or TC71 parental cells (n = 3 for each model); number of genes is indicated for each category. p values: two-tailed paired Wilcoxon test. Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. (D) Heatmap for the core set of commonly upregulated (left panel) and downregulated (right panel) genes identified in (B and C) for STAG2 KO and KD Ewing cell lines. Time after siRNA transfection is indicated at the top, sgRNA and siRNA identifiers at the bottom. See also Figures S1 and S2, Table S1.

    Journal: Cancer cell

    Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

    doi: 10.1016/j.ccell.2021.04.001

    Figure Lengend Snippet: Figure 1. STAG2 knockout profoundly alters the transcriptomic landscape (A) Representative western blotting in cellular extracts from isogenic STAG2 knockout (KO) (generated with two independent sgRNAs: SA2m#1 and SA2m#2), STAG1 KO (sgRNA: SA1m#1), STAG2 rescued (sgRNA: SA2r) and STAG2 knockdown (KD) at 48 h (generated with two independent siRNAs: siSA2#6 and siSA2#8) Ewing sarcoma cells. Color code for sgRNA isogenic models is indicated for each model and kept identical throughout the article. (B) Scaled Venn diagram for modulated genes between STAG2 WT and KO conditions (n = 3); total modulated genes for each condition represent the sum of intra- circle numbers; universe includes expressed genes (n = 13,780). p value for intersection was calculated with the SuperExact test. (C) Boxplots of log2 fold change for up-, un-, and downregulated genes in STAG2 KO and STAG2 rescued cells as compared with A673 or TC71 parental cells (n = 3 for each model); number of genes is indicated for each category. p values: two-tailed paired Wilcoxon test. Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. (D) Heatmap for the core set of commonly upregulated (left panel) and downregulated (right panel) genes identified in (B and C) for STAG2 KO and KD Ewing cell lines. Time after siRNA transfection is indicated at the top, sgRNA and siRNA identifiers at the bottom. See also Figures S1 and S2, Table S1.

    Article Snippet: The Ewing sarcoma A673 cell line was obtained from the American Type Culture Collection (ATCC) and the Ewing sarcoma TC71 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

    Techniques: Knock-Out, Western Blot, Generated, Knockdown, Two Tailed Test, Transfection

    Figure 5. STAG2 mutation is globally associated with decreased cis-promoter-enhancer and enhancer-enhancer interactions within loops (A–D) Color-coded boxplots of comparative analysis of (A–B) H3K27ac ChIP-seq peak intensities and (C–D) H3K27ac HiChIP interactions along promoter- enhancer chains between STAG2 WT and KO as well as between STAG2 KO and rescued cells. p values: two-tailed Wilcoxon test. P (promoter) and E (enhancer) positions in the chain are shown for ranks 1 to 5. (E) Color-coded boxplots of comparative analysis of intra super-enhancer interactions in H3K27ac HiChIP data between STAG2 WT and KO or rescued cells. p values: two-tailed t test. (A–E) Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. The n value for each condition is indicated. (F) Top, curve of cumulative percentage of loop presence upon genomic distance (log10 scale) in A673 and TC71 (STAG2 WT). Loop size threshold: 75% of cumulative loops corresponding to 595 kb for A673 (red line) and 340 kb (green line) for TC71. Numbers of loop and median loop size for each cell line is indicated. Bottom, percentage of cis-interaction read pairs upon genomic distance between STAG2 WT, KO, and rescued conditions in CTCF and H3K27ac HiChIP data. A threshold of 20 kb used for H3K27ac chain detection is displayed (blue dashed line). Right, zoom in CTCF and H3K27ac HiChIP plots flanking lower and top thresholds (highlighted in gray). See also Figures S7 and S8, Tables S4, S5, and S6.

    Journal: Cancer cell

    Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

    doi: 10.1016/j.ccell.2021.04.001

    Figure Lengend Snippet: Figure 5. STAG2 mutation is globally associated with decreased cis-promoter-enhancer and enhancer-enhancer interactions within loops (A–D) Color-coded boxplots of comparative analysis of (A–B) H3K27ac ChIP-seq peak intensities and (C–D) H3K27ac HiChIP interactions along promoter- enhancer chains between STAG2 WT and KO as well as between STAG2 KO and rescued cells. p values: two-tailed Wilcoxon test. P (promoter) and E (enhancer) positions in the chain are shown for ranks 1 to 5. (E) Color-coded boxplots of comparative analysis of intra super-enhancer interactions in H3K27ac HiChIP data between STAG2 WT and KO or rescued cells. p values: two-tailed t test. (A–E) Boxes represent the central 50% of data points (interquartile range). Upper and lower whiskers represent the largest and smallest observed values within 1.5 times the interquartile range from the ends of the box. The n value for each condition is indicated. (F) Top, curve of cumulative percentage of loop presence upon genomic distance (log10 scale) in A673 and TC71 (STAG2 WT). Loop size threshold: 75% of cumulative loops corresponding to 595 kb for A673 (red line) and 340 kb (green line) for TC71. Numbers of loop and median loop size for each cell line is indicated. Bottom, percentage of cis-interaction read pairs upon genomic distance between STAG2 WT, KO, and rescued conditions in CTCF and H3K27ac HiChIP data. A threshold of 20 kb used for H3K27ac chain detection is displayed (blue dashed line). Right, zoom in CTCF and H3K27ac HiChIP plots flanking lower and top thresholds (highlighted in gray). See also Figures S7 and S8, Tables S4, S5, and S6.

    Article Snippet: The Ewing sarcoma A673 cell line was obtained from the American Type Culture Collection (ATCC) and the Ewing sarcoma TC71 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

    Techniques: Mutagenesis, ChIP-sequencing, HiChIP, Two Tailed Test

    Purification and characterization of EV preparations from EwS and fibroblast cell lines and healthy donor plasma. ( A ) Size distribution of EVs isolated by ultrafiltration and differential centrifugation from healthy donor plasma, MRC5 fibroblasts, and A4573, A673, TC32, and TC71 EwS cell lines. Nanoparticle tracking analysis of three independent EV preparations (dotted line) and respective mean (solid line) is shown. ( B ) Immunoblotting-detection of chaperon proteins Hsp70 and Hsp90, EV markers CD63, CD81 and TSG101, tubulin and actin in the purified EV preparations from EwS cell lines and healthy donor plasma ( n = 3 independent donors). ( C ) Expression of the CD63 and CD81 EV markers (solid line) compared to IT antibodies (dotted line) on MRC5 EVs or EwS EVs bound to 3.9 µm latex beads. Representative flow cytometry result (a total of 3 independent EV preparations) is shown. ( D ) TNF release from CD14 + monocytes treated for 6 h with three independent EV preparations (3 × 10 9 EVs/mL) from healthy donor plasma, MRC5, or EwS cell lines. Conditioned medium was analyzed by cytokine singleplex assay.

    Journal: Cells

    Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

    doi: 10.3390/cells10082081

    Figure Lengend Snippet: Purification and characterization of EV preparations from EwS and fibroblast cell lines and healthy donor plasma. ( A ) Size distribution of EVs isolated by ultrafiltration and differential centrifugation from healthy donor plasma, MRC5 fibroblasts, and A4573, A673, TC32, and TC71 EwS cell lines. Nanoparticle tracking analysis of three independent EV preparations (dotted line) and respective mean (solid line) is shown. ( B ) Immunoblotting-detection of chaperon proteins Hsp70 and Hsp90, EV markers CD63, CD81 and TSG101, tubulin and actin in the purified EV preparations from EwS cell lines and healthy donor plasma ( n = 3 independent donors). ( C ) Expression of the CD63 and CD81 EV markers (solid line) compared to IT antibodies (dotted line) on MRC5 EVs or EwS EVs bound to 3.9 µm latex beads. Representative flow cytometry result (a total of 3 independent EV preparations) is shown. ( D ) TNF release from CD14 + monocytes treated for 6 h with three independent EV preparations (3 × 10 9 EVs/mL) from healthy donor plasma, MRC5, or EwS cell lines. Conditioned medium was analyzed by cytokine singleplex assay.

    Article Snippet: A4573, A673, TC32, and TC71 EwS cell lines and MRC5 fibroblast cell line (DSMZ; Braunschweig, Germany) were cultured in RPMI 1640 or DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (all obtained from Life Technologies, Grand Island, NY, USA).

    Techniques: Purification, Clinical Proteomics, Isolation, Centrifugation, Western Blot, Expressing, Flow Cytometry, Singleplex Assay

    EwS EVs induce pro-inflammatory responses in myeloid cells. ( A – F ) Cytokine and chemokine multiplex profiling of the conditioned medium from myeloid cells treated with PBS control (Ctrl) or EVs from plasma of healthy donors, MRC5 fibroblasts, or EwS cell lines, as indicated. ( A ) CD33 + myeloid cells and ( B ) CD14 + monocytes were treated with 3 × 10 9 EVs/mL from A4573, A673, and TC32 EwS cell lines or PBS for 24 h. ( C ) CD33 + myeloid cells were incubated for 24 h with 5 × 10 8 to 7.5 × 10 9 EVs/mL from an A4573 cell line or healthy donor plasma. ( D ) CD33 + myeloid cells were incubated with 5 × 10 8 to 7.5 × 10 9 EVs/mL from A673 EwS cells or MRC5 fibroblasts. The medium was changed after 6-h treatment and analyzed 18 h later. ( E ) CD14 + myeloid cells were incubated with 3.75 or 7 × 10 9 EVs/mL from TC32 and TC71 EwS cells or PBS. The medium was changed after 6-h treatment and analyzed 18 h later. ( F ) CD33 + myeloid cells were differentiated with the IL-4 and GM-CSF cocktail in the presence of 3 × 10 9 EVs/mL from TC32 or MRC5 cells, or PBS. Conditioned medium was analyzed on days 3 and 5. Results were obtained from one ( E ), two ( C ), or three ( A , B , D , F ) independent donors with one ( D ), two ( C ), or three ( A , B , E , F ) independent EV preparations. Data are presented as mean ± SD. An unpaired two-tailed t -test was used to calculate p values ( E ). * p ≤ 0.05.

    Journal: Cells

    Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

    doi: 10.3390/cells10082081

    Figure Lengend Snippet: EwS EVs induce pro-inflammatory responses in myeloid cells. ( A – F ) Cytokine and chemokine multiplex profiling of the conditioned medium from myeloid cells treated with PBS control (Ctrl) or EVs from plasma of healthy donors, MRC5 fibroblasts, or EwS cell lines, as indicated. ( A ) CD33 + myeloid cells and ( B ) CD14 + monocytes were treated with 3 × 10 9 EVs/mL from A4573, A673, and TC32 EwS cell lines or PBS for 24 h. ( C ) CD33 + myeloid cells were incubated for 24 h with 5 × 10 8 to 7.5 × 10 9 EVs/mL from an A4573 cell line or healthy donor plasma. ( D ) CD33 + myeloid cells were incubated with 5 × 10 8 to 7.5 × 10 9 EVs/mL from A673 EwS cells or MRC5 fibroblasts. The medium was changed after 6-h treatment and analyzed 18 h later. ( E ) CD14 + myeloid cells were incubated with 3.75 or 7 × 10 9 EVs/mL from TC32 and TC71 EwS cells or PBS. The medium was changed after 6-h treatment and analyzed 18 h later. ( F ) CD33 + myeloid cells were differentiated with the IL-4 and GM-CSF cocktail in the presence of 3 × 10 9 EVs/mL from TC32 or MRC5 cells, or PBS. Conditioned medium was analyzed on days 3 and 5. Results were obtained from one ( E ), two ( C ), or three ( A , B , D , F ) independent donors with one ( D ), two ( C ), or three ( A , B , E , F ) independent EV preparations. Data are presented as mean ± SD. An unpaired two-tailed t -test was used to calculate p values ( E ). * p ≤ 0.05.

    Article Snippet: A4573, A673, TC32, and TC71 EwS cell lines and MRC5 fibroblast cell line (DSMZ; Braunschweig, Germany) were cultured in RPMI 1640 or DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (all obtained from Life Technologies, Grand Island, NY, USA).

    Techniques: Multiplex Assay, Control, Clinical Proteomics, Incubation, Two Tailed Test

    EwS EVs affect myeloid cells at an early stage of differentiation and impair their maturation. ( A ) Experimental design ( B – D ). CD14 + or CD33 + myeloid cells purified from healthy donors were differentiated in the presence of GM-CSF and IL-4 for 5 days and matured with the IL-1β, IL-6, PGE 2 , and TNF cocktail for additional 2 days to moDCs. The indicated EVs (3 × 10 9 /mL) or PBS control (Ctrl) were added at day 0 of differentiation. ( B – E ) Flow cytometry analysis of the indicated maturation markers at day 7. ( B ) Geometric mean fluorescence intensity (gMFI) of CD80, CD86, and HLA-DR normalized to IT of CD14 + monocytes treated with EVs from MRC5, A673, TC32, or TC71 cells, or PBS. Fold change to Ctrl is shown. ( C ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD14 + myeloid cells treated with three independent EV preparations from TC32 cells or PBS. ( D ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD33 + myeloid cells treated with MRC5 EVs, TC32 EVs, or PBS. ( E ) TC32 EVs were added to CD14 + or CD33 + myeloid cells during differentiation (day 0 and 3) or maturation (day 5). Representative pseudocolor plots of CD80, CD83, CD86, and HLA-DR compared to the respective IT antibodies are shown. The results were obtained from one ( C ) or three ( B , D , E ) independent donors with three ( B – E ) independent EV preparations. Data are presented as mean ± SD. One-way ANOVA with multiple comparison Turkey test ( B ), and unpaired ( C ) and paired ( D ) two-tailed t -tests were used to calculate p values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001.

    Journal: Cells

    Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

    doi: 10.3390/cells10082081

    Figure Lengend Snippet: EwS EVs affect myeloid cells at an early stage of differentiation and impair their maturation. ( A ) Experimental design ( B – D ). CD14 + or CD33 + myeloid cells purified from healthy donors were differentiated in the presence of GM-CSF and IL-4 for 5 days and matured with the IL-1β, IL-6, PGE 2 , and TNF cocktail for additional 2 days to moDCs. The indicated EVs (3 × 10 9 /mL) or PBS control (Ctrl) were added at day 0 of differentiation. ( B – E ) Flow cytometry analysis of the indicated maturation markers at day 7. ( B ) Geometric mean fluorescence intensity (gMFI) of CD80, CD86, and HLA-DR normalized to IT of CD14 + monocytes treated with EVs from MRC5, A673, TC32, or TC71 cells, or PBS. Fold change to Ctrl is shown. ( C ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD14 + myeloid cells treated with three independent EV preparations from TC32 cells or PBS. ( D ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD33 + myeloid cells treated with MRC5 EVs, TC32 EVs, or PBS. ( E ) TC32 EVs were added to CD14 + or CD33 + myeloid cells during differentiation (day 0 and 3) or maturation (day 5). Representative pseudocolor plots of CD80, CD83, CD86, and HLA-DR compared to the respective IT antibodies are shown. The results were obtained from one ( C ) or three ( B , D , E ) independent donors with three ( B – E ) independent EV preparations. Data are presented as mean ± SD. One-way ANOVA with multiple comparison Turkey test ( B ), and unpaired ( C ) and paired ( D ) two-tailed t -tests were used to calculate p values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001.

    Article Snippet: A4573, A673, TC32, and TC71 EwS cell lines and MRC5 fibroblast cell line (DSMZ; Braunschweig, Germany) were cultured in RPMI 1640 or DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (all obtained from Life Technologies, Grand Island, NY, USA).

    Techniques: Purification, Control, Flow Cytometry, Fluorescence, Comparison, Two Tailed Test

    EwS EVs rewire gene expression programs associated with inflammatory responses and the maturation of myeloid cells. Experimental design ( A ) and whole transcriptome analysis ( B – E ). CD14 + monocytes were differentiated with the GM-CSF and IL-4 cocktail for 4 days in the presence of EVs from TC32 or TC71 EwS cells, or PBS (control), and subjected to RNA-seq. ( B ) Venn diagram showing an overlap between the sets of expressed genes (≥10 FPKM) in EV-treated and control cells. ( C ) CIBSESORT analysis of the identified mRNAs. The plot shows the four most represented immune cell sub-populations in EV-treated and control cells. ( D , E ) Heatmaps of the M1, M2, and DC ( D ) and IFN-type I/III ( E ) signature genes using the customized MSigDB Hallmark gene sets. Data represent expression values sorted according to fold change in TC32 and TC71 EV-treated CD14 + cells compared to control. Visualizations were done using FPKM values generated with cufflinks, scaled, and centered prior to plotting. Genes shown in ( D ) are grouped in the categories cytokines ( blue ), surface molecules ( red ), regulatory proteins ( green ), and toll-like receptors (TLR, pink ).

    Journal: Cells

    Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

    doi: 10.3390/cells10082081

    Figure Lengend Snippet: EwS EVs rewire gene expression programs associated with inflammatory responses and the maturation of myeloid cells. Experimental design ( A ) and whole transcriptome analysis ( B – E ). CD14 + monocytes were differentiated with the GM-CSF and IL-4 cocktail for 4 days in the presence of EVs from TC32 or TC71 EwS cells, or PBS (control), and subjected to RNA-seq. ( B ) Venn diagram showing an overlap between the sets of expressed genes (≥10 FPKM) in EV-treated and control cells. ( C ) CIBSESORT analysis of the identified mRNAs. The plot shows the four most represented immune cell sub-populations in EV-treated and control cells. ( D , E ) Heatmaps of the M1, M2, and DC ( D ) and IFN-type I/III ( E ) signature genes using the customized MSigDB Hallmark gene sets. Data represent expression values sorted according to fold change in TC32 and TC71 EV-treated CD14 + cells compared to control. Visualizations were done using FPKM values generated with cufflinks, scaled, and centered prior to plotting. Genes shown in ( D ) are grouped in the categories cytokines ( blue ), surface molecules ( red ), regulatory proteins ( green ), and toll-like receptors (TLR, pink ).

    Article Snippet: A4573, A673, TC32, and TC71 EwS cell lines and MRC5 fibroblast cell line (DSMZ; Braunschweig, Germany) were cultured in RPMI 1640 or DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (all obtained from Life Technologies, Grand Island, NY, USA).

    Techniques: Gene Expression, Control, RNA Sequencing, Expressing, Generated

    Antitumor activity of SST0001 against TC71 Ewing’s sarcoma

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

    doi: 10.1111/j.1582-4934.2010.01190.x

    Figure Lengend Snippet: Antitumor activity of SST0001 against TC71 Ewing’s sarcoma

    Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

    Techniques: Activity Assay

    Heparanase staining in Ewing’s sarcoma patients

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

    doi: 10.1111/j.1582-4934.2010.01190.x

    Figure Lengend Snippet: Heparanase staining in Ewing’s sarcoma patients

    Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

    Techniques: Staining

    Immunohistochemical staining of heparanase in Ewing’s sarcoma specimens. Formalin-fixed, paraffin-embedded 5 μm sections were subjected to immunostaining, applying anti-heparanase polyclonal antibody (Ab 733), as described under ‘Materials and methods’. Shown are representative photomicrographs of heparanase-positive specimens categorized as weak (+1, A, B) or strong (+2, C, D) intensity. Nuclear localization of heparanase is shown in (E). Specimens that were similarly stained with pre-immune serum, or applying the above procedure but lacking the primary antibody, yielded no detectable staining (F). Original magnification: (A, C) 100×; (B, D–F) 400×.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

    doi: 10.1111/j.1582-4934.2010.01190.x

    Figure Lengend Snippet: Immunohistochemical staining of heparanase in Ewing’s sarcoma specimens. Formalin-fixed, paraffin-embedded 5 μm sections were subjected to immunostaining, applying anti-heparanase polyclonal antibody (Ab 733), as described under ‘Materials and methods’. Shown are representative photomicrographs of heparanase-positive specimens categorized as weak (+1, A, B) or strong (+2, C, D) intensity. Nuclear localization of heparanase is shown in (E). Specimens that were similarly stained with pre-immune serum, or applying the above procedure but lacking the primary antibody, yielded no detectable staining (F). Original magnification: (A, C) 100×; (B, D–F) 400×.

    Article Snippet: The Ewing’s sarcoma cell line TC71 was maintained in Iscove’s modified Dulbecco’s Medium (Lonza, Verviers, Belgium) supplemented with 10% foetal bovine serum at 37°C in 5% CO 2 atmosphere.

    Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Immunostaining