ss18 ssx fusion site protein  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ss18 ssx fusion site protein
    Ss18 Ssx Fusion Site Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ss18 ssx fusion site protein/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    ss18 ssx fusion site protein - by Bioz Stars, 2023-02
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    ss18 ssx fusion site protein  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ss18 ssx fusion site protein
    Ss18 Ssx Fusion Site Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    antibodies h2ak119ub  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies h2ak119ub
    a) Left, Immunofluorescence against BCOR and <t>H2AK119ub</t> in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.
    Antibodies H2ak119ub, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies h2ak119ub/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies h2ak119ub - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma"

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    Journal: bioRxiv

    doi: 10.1101/2022.07.18.499263

    a) Left, Immunofluorescence against BCOR and H2AK119ub in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.
    Figure Legend Snippet: a) Left, Immunofluorescence against BCOR and H2AK119ub in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.

    Techniques Used: Immunofluorescence, Expressing, Construct, Staining, Fluorescence, One-tailed Test, Quantitative RT-PCR, Western Blot, Marker

    a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).
    Figure Legend Snippet: a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Techniques Used: Knock-In, Expressing, Immunofluorescence, Staining, One-tailed Test, Microarray, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).
    Figure Legend Snippet: a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Techniques Used: Immunofluorescence, Expressing, Staining, One-tailed Test

    ssx ss18  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ssx ss18
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
    Ssx Ss18, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ssx ss18/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ssx ss18 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma"

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    Journal: bioRxiv

    doi: 10.1101/2022.07.18.499263

    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in SS18-SSX1 using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, SSX-C (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
    Figure Legend Snippet: a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in SS18-SSX1 using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, SSX-C (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).

    Techniques Used: CRISPR, Knock-Out, Sequencing, Construct, Imaging, Expressing, One-tailed Test, Quantitative RT-PCR, Plasmid Preparation

    a), b) CRISPR knock-out hypersensitive (CKHS) regions and PFAM domain annotation for SS18 (a) and SSX1 (b) in control, fusion negative cell line (KHOS-240S, osteosarcoma cell line) and in HS-SY-II synovial sarcoma cell line harbouring an SS18-SSX1 fusion. CKHS regions are highlighted in dark red. c), d) Live confocal imaging images of metaphase HEK293T expressing eGFP fused constructs for SS18, SS18-SSX1, SSX-C, SSXRDand SSX-C ΔRD (c)orKDM2B, PCGF1 and RING1B (d). DNA is stained using Hoechst 33342. Scale bars correspond to 5μm.
    Figure Legend Snippet: a), b) CRISPR knock-out hypersensitive (CKHS) regions and PFAM domain annotation for SS18 (a) and SSX1 (b) in control, fusion negative cell line (KHOS-240S, osteosarcoma cell line) and in HS-SY-II synovial sarcoma cell line harbouring an SS18-SSX1 fusion. CKHS regions are highlighted in dark red. c), d) Live confocal imaging images of metaphase HEK293T expressing eGFP fused constructs for SS18, SS18-SSX1, SSX-C, SSXRDand SSX-C ΔRD (c)orKDM2B, PCGF1 and RING1B (d). DNA is stained using Hoechst 33342. Scale bars correspond to 5μm.

    Techniques Used: CRISPR, Knock-Out, Imaging, Expressing, Construct, Staining

    a) Schematic representing the layout of the eGFP pull down and mass-spectrometry analysis. Common SSX-C and SSXRD enriched hits (highlighted in orange) were identified. b) Log2 fold change correlation plot of eGFP-SSXRD and eGFP-SSX-C mass spectrometry data following eGFP pull down in HS-SY-II cells. Data was normalized to eGFP-SSX-C ΔRD . c) Log fold change plot between eGFP-SSX-C and eGFP-SSX-C E184 * mass spectrometry data following eGFP pull down in two biological replicates. Data was normalized to eGFP. d) BRET ratio (mBU) in Nluc-H2A or Nluc-H2A K118K119R transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups ** p< 0.01, * p< 0.05. e) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A or Nluc-H2A K118K119R revealed with NLuc, H2AK119ub1 and H3 antibodies. f) Illustration of the two MacroH2A genes, H2AFY encoding the two isoforms MacroH2A1.1 and MacroH2A1.2 which differ by one exon (grey/white box) within the Macro domain (pink) and H2AFY2 encoding MacroH2A2. g) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A, Nluc-MacroH2A1.2 or Nluc-MacroH2A2. Detection was performed using NLuc mixed with H2AK119ub1 and H3 antibodies. h) BRET ratio (mBU) in Nluc-H2A, Nluc-macroH2A1.2 or Nluc-macroH2A2 transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 4 biological replicates. Asterisks represent p-values of paired onetailed t-test between groups (* p< 0.05, ** p< 0.01). i) Heatmaps of HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II cells (n=52027). Rows correspond to ±5-kb regions across the midpoint of each signal, ranked by increasing signal. j) Gene tracks for HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN in HS-SY-II cells at the MNX1, KCNQ2, UNCX and SOX8 .
    Figure Legend Snippet: a) Schematic representing the layout of the eGFP pull down and mass-spectrometry analysis. Common SSX-C and SSXRD enriched hits (highlighted in orange) were identified. b) Log2 fold change correlation plot of eGFP-SSXRD and eGFP-SSX-C mass spectrometry data following eGFP pull down in HS-SY-II cells. Data was normalized to eGFP-SSX-C ΔRD . c) Log fold change plot between eGFP-SSX-C and eGFP-SSX-C E184 * mass spectrometry data following eGFP pull down in two biological replicates. Data was normalized to eGFP. d) BRET ratio (mBU) in Nluc-H2A or Nluc-H2A K118K119R transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups ** p< 0.01, * p< 0.05. e) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A or Nluc-H2A K118K119R revealed with NLuc, H2AK119ub1 and H3 antibodies. f) Illustration of the two MacroH2A genes, H2AFY encoding the two isoforms MacroH2A1.1 and MacroH2A1.2 which differ by one exon (grey/white box) within the Macro domain (pink) and H2AFY2 encoding MacroH2A2. g) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A, Nluc-MacroH2A1.2 or Nluc-MacroH2A2. Detection was performed using NLuc mixed with H2AK119ub1 and H3 antibodies. h) BRET ratio (mBU) in Nluc-H2A, Nluc-macroH2A1.2 or Nluc-macroH2A2 transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 4 biological replicates. Asterisks represent p-values of paired onetailed t-test between groups (* p< 0.05, ** p< 0.01). i) Heatmaps of HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II cells (n=52027). Rows correspond to ±5-kb regions across the midpoint of each signal, ranked by increasing signal. j) Gene tracks for HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN in HS-SY-II cells at the MNX1, KCNQ2, UNCX and SOX8 .

    Techniques Used: Mass Spectrometry, Transfection, Expressing, Plasmid Preparation, One-tailed Test, Western Blot

    a) Up, Representation of the methyl binding domain (MBD)-mediated targeting approach of proteins to methylated CpG. Here, the CXXC mutated KDM2B is redirected to methylated CpG via the MBD. Bottom, Schematic representing the MBD (black square) fusion constructs for Luciferase control (Luc) and KDM2B. The KDM2B long isoform contains the histone demethylase JmjC domain (gold box) and a mutated CXXC domain (dark orange). Both constructs contain a V5 tag. b) Left, Immunofluorescence of human synovial sarcoma (HS-SY-II) cells displaying the MBD constructs (V5, magenta), BCOR (green) and H2AK119ub1 (cyan). Yellow arrow heads point to the MBD foci. Scale bars represents 5μm throughout the figure. Right, quantification of the percentage of BCOR or H2AK119ub1 foci overlapping a V5 foci in 3 or 4 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Left, Immunofluorescence for V5 (magenta) and SS18-SSX1 (HA, cyan). Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 5 biological replicates. d) Left, Immunofluorescence for V5 (magenta) and H3K27me3 (cyan). Right, quantification of the percentage of BCOR or H3K27me3 foci overlapping with V5 foci in 2 biological replicates. Data represents the mean and S.E.M. e) Left, Immunofluorescence of MBD-KDM2B (V5, magenta) in the presence of different sgRNAs (eGFP background fluorescence) with SS18-SSX1 (HA, cyan) in in HS-SY-II-Cas9 cells. Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 4 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (** p< 0.01 and *** p< 0.001). f) Left, Immunofluorescence images of MBD-KDM2B (V5, magenta) with SS18-SSX or H2AK119ub1 or MacroH2A1.1 (cyan) in HS-SY-II-Cas9 cells expressing sgCtrl, sgMacroH2A (targeting both histone genes H2AFY and H2AFY2) or sgPCGFI. Right, quantification of the percentage of foci overlapping MBD-KDM2B foci in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (* p< 0.05 and ** p< 0.01).
    Figure Legend Snippet: a) Up, Representation of the methyl binding domain (MBD)-mediated targeting approach of proteins to methylated CpG. Here, the CXXC mutated KDM2B is redirected to methylated CpG via the MBD. Bottom, Schematic representing the MBD (black square) fusion constructs for Luciferase control (Luc) and KDM2B. The KDM2B long isoform contains the histone demethylase JmjC domain (gold box) and a mutated CXXC domain (dark orange). Both constructs contain a V5 tag. b) Left, Immunofluorescence of human synovial sarcoma (HS-SY-II) cells displaying the MBD constructs (V5, magenta), BCOR (green) and H2AK119ub1 (cyan). Yellow arrow heads point to the MBD foci. Scale bars represents 5μm throughout the figure. Right, quantification of the percentage of BCOR or H2AK119ub1 foci overlapping a V5 foci in 3 or 4 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Left, Immunofluorescence for V5 (magenta) and SS18-SSX1 (HA, cyan). Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 5 biological replicates. d) Left, Immunofluorescence for V5 (magenta) and H3K27me3 (cyan). Right, quantification of the percentage of BCOR or H3K27me3 foci overlapping with V5 foci in 2 biological replicates. Data represents the mean and S.E.M. e) Left, Immunofluorescence of MBD-KDM2B (V5, magenta) in the presence of different sgRNAs (eGFP background fluorescence) with SS18-SSX1 (HA, cyan) in in HS-SY-II-Cas9 cells. Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 4 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (** p< 0.01 and *** p< 0.001). f) Left, Immunofluorescence images of MBD-KDM2B (V5, magenta) with SS18-SSX or H2AK119ub1 or MacroH2A1.1 (cyan) in HS-SY-II-Cas9 cells expressing sgCtrl, sgMacroH2A (targeting both histone genes H2AFY and H2AFY2) or sgPCGFI. Right, quantification of the percentage of foci overlapping MBD-KDM2B foci in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (* p< 0.05 and ** p< 0.01).

    Techniques Used: Binding Assay, Methylation, Construct, Luciferase, Immunofluorescence, Fluorescence, One-tailed Test, Expressing

    a) Heatmaps of H2AK119ub1 (purple) or SS18-SSX (blue) CUT&RUN signals in HS-SY-II (left) and SYO-I (right) Cas9 cells expressing empty sgRNA as control (EV) or targeting PCGF1 (sgPCGFI). Both heatmaps represent CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or SS18-SSX2 peaks in SYO-I (right, n=61940). Rows correspond to ±5-kb regions across the midpoint of each enriched region, ranked by increasing signal. b) H2AK119ub1 and SS18-SSX CUT&RUN score distributions across HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or the SS18-SSX2 peaks in SYO-I (right, n=61940). c) Gene track for H2AK119ub1 and SS18-SSX CUT&RUN at the EN2-SHH-NOM1 and FGF4-FGF3 loci. d) Salt extraction assay displaying SS18-SSX1 levels by western blot in HS-SY-II-Cas9 cells expressing an empty vector (EV) or sgRNAs against PCGF1 or PCGF3. e) Quantification of the SS18-SSX protein distribution in the various salt extraction fractions. Data represents the mean and S.E.M for the percentage of total protein per fraction in 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05).
    Figure Legend Snippet: a) Heatmaps of H2AK119ub1 (purple) or SS18-SSX (blue) CUT&RUN signals in HS-SY-II (left) and SYO-I (right) Cas9 cells expressing empty sgRNA as control (EV) or targeting PCGF1 (sgPCGFI). Both heatmaps represent CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or SS18-SSX2 peaks in SYO-I (right, n=61940). Rows correspond to ±5-kb regions across the midpoint of each enriched region, ranked by increasing signal. b) H2AK119ub1 and SS18-SSX CUT&RUN score distributions across HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or the SS18-SSX2 peaks in SYO-I (right, n=61940). c) Gene track for H2AK119ub1 and SS18-SSX CUT&RUN at the EN2-SHH-NOM1 and FGF4-FGF3 loci. d) Salt extraction assay displaying SS18-SSX1 levels by western blot in HS-SY-II-Cas9 cells expressing an empty vector (EV) or sgRNAs against PCGF1 or PCGF3. e) Quantification of the SS18-SSX protein distribution in the various salt extraction fractions. Data represents the mean and S.E.M for the percentage of total protein per fraction in 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05).

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, One-tailed Test

    a) Log2 Fold change of RPKM values from RNA sequencing in CRL7250 cells expressing SS18 or SS18-SSX1 compared to naive cells in two biological replicates. Data from (McBride et al., 2018). b) Log2 Fold change of RPKM values from RNA sequencing in HS-SY-II and SYO-I cells after knockdown of SS18-SSX compared to shCtrl cells in two biological replicates. Data from (McBride et al., 2018). c, d) Western blot of whole cell extracts from mesenchymal stem cells expressing eGFP or eGFP-SS18-SSX1 (c); and from HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI) (d). Proteins were detected using BCOR and PCGF1 antibodies and Beta-actin was used as loading control. e) Gene tracks for HA-SS18-SSX1 and SS18-SSX2 CUT&RUN at the BCOR and RYBP loci. f) Left, Immunofluorescence of eGFP-fused constructs (eGFP, eGFP-SS18-SSX1) expressed in HS-SY-II with eGFP signals (green) and nucleus stained with DAPI and H2AK119ub1. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). g) Left, H2AK119ub1 immunofluorescence in HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI). sgRNA expressing cells are positive for eGFP. Cells were mixed with non sgRNA expressing cells for direct comparison of H2AK119ub1 signal intensity (yellow arrow heads). Right, quantification of the H2AK119ub1 fluorescence ratio in eGFP (=sgRNA) versus no eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars represents 10μm. Scale bars represents 20μm.
    Figure Legend Snippet: a) Log2 Fold change of RPKM values from RNA sequencing in CRL7250 cells expressing SS18 or SS18-SSX1 compared to naive cells in two biological replicates. Data from (McBride et al., 2018). b) Log2 Fold change of RPKM values from RNA sequencing in HS-SY-II and SYO-I cells after knockdown of SS18-SSX compared to shCtrl cells in two biological replicates. Data from (McBride et al., 2018). c, d) Western blot of whole cell extracts from mesenchymal stem cells expressing eGFP or eGFP-SS18-SSX1 (c); and from HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI) (d). Proteins were detected using BCOR and PCGF1 antibodies and Beta-actin was used as loading control. e) Gene tracks for HA-SS18-SSX1 and SS18-SSX2 CUT&RUN at the BCOR and RYBP loci. f) Left, Immunofluorescence of eGFP-fused constructs (eGFP, eGFP-SS18-SSX1) expressed in HS-SY-II with eGFP signals (green) and nucleus stained with DAPI and H2AK119ub1. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). g) Left, H2AK119ub1 immunofluorescence in HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI). sgRNA expressing cells are positive for eGFP. Cells were mixed with non sgRNA expressing cells for direct comparison of H2AK119ub1 signal intensity (yellow arrow heads). Right, quantification of the H2AK119ub1 fluorescence ratio in eGFP (=sgRNA) versus no eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars represents 10μm. Scale bars represents 20μm.

    Techniques Used: RNA Sequencing Assay, Expressing, Western Blot, Plasmid Preparation, Immunofluorescence, Construct, Staining, Fluorescence, One-tailed Test

    a) Left, Immunofluorescence against BCOR in HS-SY-II synovial sarcoma cells expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ). Right, quantification of BCOR fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. b) Sequential chromatin washes assay using 150mM salt buffer in untransduced control (Ctrl) or eGFP-SSX-C expressing HEK293T cells. BCOR, PCGF1 or Beta-Actin as a loading control were detected by western blot. c) Quantification of the protein distribution for BCOR, PCGF1 or Beta-Actin in the various washes. Data represents the percentage of total protein levels. d) Left, Immunofluorescence against H2AK119ub1 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 7 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (*** p< 0.001). Scale bars represents 20μm. e) Left, Immunofluorescence against SS18 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of the SS18 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. f) H&E and immunohistochemical staining for Inhibin-a, SSX and H2AK119ub1 in human testis. Scale bar correspond to 40μm in the upper panel. Lower panel is a close-up from the images shown in the upper panel (area marked by a dashed line) where the scale bar corresponds to 20μm.
    Figure Legend Snippet: a) Left, Immunofluorescence against BCOR in HS-SY-II synovial sarcoma cells expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ). Right, quantification of BCOR fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. b) Sequential chromatin washes assay using 150mM salt buffer in untransduced control (Ctrl) or eGFP-SSX-C expressing HEK293T cells. BCOR, PCGF1 or Beta-Actin as a loading control were detected by western blot. c) Quantification of the protein distribution for BCOR, PCGF1 or Beta-Actin in the various washes. Data represents the percentage of total protein levels. d) Left, Immunofluorescence against H2AK119ub1 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 7 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (*** p< 0.001). Scale bars represents 20μm. e) Left, Immunofluorescence against SS18 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of the SS18 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. f) H&E and immunohistochemical staining for Inhibin-a, SSX and H2AK119ub1 in human testis. Scale bar correspond to 40μm in the upper panel. Lower panel is a close-up from the images shown in the upper panel (area marked by a dashed line) where the scale bar corresponds to 20μm.

    Techniques Used: Immunofluorescence, Expressing, Construct, Fluorescence, One-tailed Test, Western Blot, Immunohistochemical staining, Staining

    a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).
    Figure Legend Snippet: a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Techniques Used: Knock-In, Expressing, Immunofluorescence, Staining, One-tailed Test, Microarray, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).
    Figure Legend Snippet: a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Techniques Used: Immunofluorescence, Expressing, Staining, One-tailed Test

    Model depicting the strong interplay between SS18-SSX and H2AK119ub1 where SS18-SSX interacts with histones H2A which are ubiquitinated on their lysine K119. SS18-SSX then promotes further levels of H2AK119ub1 via two distinct mechanisms: 1) by stimulating the transcription of PRC1.1 members BCOR and RYBP as direct targets of the fusion and 2) by increasing the stability of the PRC1.1 complex on chromatin. In both cases H2AK119ub1 levels increase and therefore reinforce SS18-SSX’s presence on chromatin.
    Figure Legend Snippet: Model depicting the strong interplay between SS18-SSX and H2AK119ub1 where SS18-SSX interacts with histones H2A which are ubiquitinated on their lysine K119. SS18-SSX then promotes further levels of H2AK119ub1 via two distinct mechanisms: 1) by stimulating the transcription of PRC1.1 members BCOR and RYBP as direct targets of the fusion and 2) by increasing the stability of the PRC1.1 complex on chromatin. In both cases H2AK119ub1 levels increase and therefore reinforce SS18-SSX’s presence on chromatin.

    Techniques Used:

    ss18 ssx  (Cell Signaling Technology Inc)


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    e9x9v  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc e9x9v
    E9x9v, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ss18 ssx fusion site protein
    Ss18 Ssx Fusion Site Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies h2ak119ub
    a) Left, Immunofluorescence against BCOR and <t>H2AK119ub</t> in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.
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    Cell Signaling Technology Inc ssx ss18
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
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    Cell Signaling Technology Inc ss18 ssx
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
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    Cell Signaling Technology Inc ss18 ssx antibody
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
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    Cell Signaling Technology Inc e9x9v
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
    E9x9v, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti ss18 ssx
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
    Anti Ss18 Ssx, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ss18 ssx fusion specific rabbit monoclonal antibody
    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in <t>SS18-SSX1</t> using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, <t>SSX-C</t> (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).
    Ss18 Ssx Fusion Specific Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a) Left, Immunofluorescence against BCOR and H2AK119ub in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Left, Immunofluorescence against BCOR and H2AK119ub in mesenchymal stem cells (MSCs) expressing the indicated eGFP fused constructs with eGFP signals and nucleus stained with DAPI (grayscale). Right, quantification of BCOR or H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 10μm throughout the panel. b) qRT-PCR displaying Log2 fold change of mRNA levels normalized by GAPDH in MSCs expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ) relative to eGFP expressing cells in two biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Salt extraction assay in HS-SY-II expressing eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD . Proteins were detected by western blot using with BCOR, PCGF1 or Beta-actin (loading control) antibodies. c) Quantification of the protein distribution in the various fractions of the salt extraction for BCOR or PCGF1. Data represents the percentage of total protein levels. d) Immunofluorescence against H2AK119ub in HS-SY-II (left) or SYO-I (right) cells expressing the indicated eGFP-fused constructs (eGFP-SSX-C and eGFP-SSX-C E184 *). On the right of each panel of IF images are quantifications of the H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01) e) Left, tSNE and clustering analysis of combined single-cell transcriptome data from human testes (n = 6490) from (Guo et al., 2018). Each dot represents a single cell and is colored according to its cluster identity as indicated on the figure key. The 13 cluster identities were assigned based on marker gene expression. Right, SSX1 expression pattern projected on the tSNE plot. Red indicates high expression and gray indicates low or no expression.

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Immunofluorescence, Expressing, Construct, Staining, Fluorescence, One-tailed Test, Quantitative RT-PCR, Western Blot, Marker

    a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Knock-In, Expressing, Immunofluorescence, Staining, One-tailed Test, Microarray, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Immunofluorescence, Expressing, Staining, One-tailed Test

    a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in SS18-SSX1 using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, SSX-C (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a ) Layout of CRISPR-Cas9 knockout gene-tilling screen. b) Mapping of CRISPR knockout hyper-sensitive (CKHS) regions in SS18-SSX1 using ProTiler based on Iog2 fold changes (LFC) of sgRNAs representation in HS-SY-II synovial sarcoma cells. The CKHS region is highlighted in dark red and corresponds to the SSXRD PFAM sequence (PF09514). c) Schematic representation of eGFP (green) fused constructs for SS18, SS18-SSX1, SSX-C (78aa of SSX1 present in the SS18-SSX1 fusion), SSXRD (last 34aa of SSX-C) or SSX-CΔRD (SSX-C with a deletion of the SSXRD). d) Live confocal imaging of the eGFP-fused constructs in HEK193T cells. Scale bar corresponds to 20μm. e) Salt extraction assay in HEK293T expressing the various eGFP constructs. The proteins are detected using an eGFP antibody. f) Percentage of total protein levels per fraction in two or three biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups, * p< 0.05. g) Heatmaps for HA-SS18-SSX1, KDM2B ChlP-seq from Banito et al., 2018 and eGFP ChIP in HS-SY-II cells expressing eGFP fused SSX-C or SSX-C ΔRD . Heatmaps represent ChlP-seq signals over HA-SS18-SSX1 broad peaks (n=26805). Rows correspond to ±5-kb regions across the midpoint of each HA-enriched region, ranked by increasing signal in HS-SY-II cells. h) Gene tracks for HA-SS18-SSX1, KDM2B and eGFP ChlP-seq at the EN2, WNT7B and KNCQ2 loci. i) Schematic representing new synovial sarcoma fusions. SS18-SSX1 and the SSX-C contain the canonical breakpoint “a”, while EWSR1-SSX1 and MN1-SSX1 exhibit an alternative breakpoint “b”. j) qRT-PCR displaying Log2 fold change of mRNA levels relative to GAPDH in mesenchymal stem cells (MSCs) expressing the new fusion constructs and controls. Values are normalized to empty vector expression in three biological replicates. Data represents the mean and the standard error of the mean (S.E.M).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: CRISPR, Knock-Out, Sequencing, Construct, Imaging, Expressing, One-tailed Test, Quantitative RT-PCR, Plasmid Preparation

    a), b) CRISPR knock-out hypersensitive (CKHS) regions and PFAM domain annotation for SS18 (a) and SSX1 (b) in control, fusion negative cell line (KHOS-240S, osteosarcoma cell line) and in HS-SY-II synovial sarcoma cell line harbouring an SS18-SSX1 fusion. CKHS regions are highlighted in dark red. c), d) Live confocal imaging images of metaphase HEK293T expressing eGFP fused constructs for SS18, SS18-SSX1, SSX-C, SSXRDand SSX-C ΔRD (c)orKDM2B, PCGF1 and RING1B (d). DNA is stained using Hoechst 33342. Scale bars correspond to 5μm.

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a), b) CRISPR knock-out hypersensitive (CKHS) regions and PFAM domain annotation for SS18 (a) and SSX1 (b) in control, fusion negative cell line (KHOS-240S, osteosarcoma cell line) and in HS-SY-II synovial sarcoma cell line harbouring an SS18-SSX1 fusion. CKHS regions are highlighted in dark red. c), d) Live confocal imaging images of metaphase HEK293T expressing eGFP fused constructs for SS18, SS18-SSX1, SSX-C, SSXRDand SSX-C ΔRD (c)orKDM2B, PCGF1 and RING1B (d). DNA is stained using Hoechst 33342. Scale bars correspond to 5μm.

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: CRISPR, Knock-Out, Imaging, Expressing, Construct, Staining

    a) Schematic representing the layout of the eGFP pull down and mass-spectrometry analysis. Common SSX-C and SSXRD enriched hits (highlighted in orange) were identified. b) Log2 fold change correlation plot of eGFP-SSXRD and eGFP-SSX-C mass spectrometry data following eGFP pull down in HS-SY-II cells. Data was normalized to eGFP-SSX-C ΔRD . c) Log fold change plot between eGFP-SSX-C and eGFP-SSX-C E184 * mass spectrometry data following eGFP pull down in two biological replicates. Data was normalized to eGFP. d) BRET ratio (mBU) in Nluc-H2A or Nluc-H2A K118K119R transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups ** p< 0.01, * p< 0.05. e) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A or Nluc-H2A K118K119R revealed with NLuc, H2AK119ub1 and H3 antibodies. f) Illustration of the two MacroH2A genes, H2AFY encoding the two isoforms MacroH2A1.1 and MacroH2A1.2 which differ by one exon (grey/white box) within the Macro domain (pink) and H2AFY2 encoding MacroH2A2. g) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A, Nluc-MacroH2A1.2 or Nluc-MacroH2A2. Detection was performed using NLuc mixed with H2AK119ub1 and H3 antibodies. h) BRET ratio (mBU) in Nluc-H2A, Nluc-macroH2A1.2 or Nluc-macroH2A2 transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 4 biological replicates. Asterisks represent p-values of paired onetailed t-test between groups (* p< 0.05, ** p< 0.01). i) Heatmaps of HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II cells (n=52027). Rows correspond to ±5-kb regions across the midpoint of each signal, ranked by increasing signal. j) Gene tracks for HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN in HS-SY-II cells at the MNX1, KCNQ2, UNCX and SOX8 .

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Schematic representing the layout of the eGFP pull down and mass-spectrometry analysis. Common SSX-C and SSXRD enriched hits (highlighted in orange) were identified. b) Log2 fold change correlation plot of eGFP-SSXRD and eGFP-SSX-C mass spectrometry data following eGFP pull down in HS-SY-II cells. Data was normalized to eGFP-SSX-C ΔRD . c) Log fold change plot between eGFP-SSX-C and eGFP-SSX-C E184 * mass spectrometry data following eGFP pull down in two biological replicates. Data was normalized to eGFP. d) BRET ratio (mBU) in Nluc-H2A or Nluc-H2A K118K119R transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups ** p< 0.01, * p< 0.05. e) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A or Nluc-H2A K118K119R revealed with NLuc, H2AK119ub1 and H3 antibodies. f) Illustration of the two MacroH2A genes, H2AFY encoding the two isoforms MacroH2A1.1 and MacroH2A1.2 which differ by one exon (grey/white box) within the Macro domain (pink) and H2AFY2 encoding MacroH2A2. g) Western blot of histone acid extracts from HEK293T cells transfected with either Nluc-H2A, Nluc-MacroH2A1.2 or Nluc-MacroH2A2. Detection was performed using NLuc mixed with H2AK119ub1 and H3 antibodies. h) BRET ratio (mBU) in Nluc-H2A, Nluc-macroH2A1.2 or Nluc-macroH2A2 transfected HEK293T cells expressing empty vector HALO, HALO-SSX-C, HALO-SSX-C ΔRD or HALO-SSX-C E184 *. Values represent 4 biological replicates. Asterisks represent p-values of paired onetailed t-test between groups (* p< 0.05, ** p< 0.01). i) Heatmaps of HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II cells (n=52027). Rows correspond to ±5-kb regions across the midpoint of each signal, ranked by increasing signal. j) Gene tracks for HA-SS18-SSX1, H2AK119ub1 and MacroH2A2 CUT&RUN in HS-SY-II cells at the MNX1, KCNQ2, UNCX and SOX8 .

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Mass Spectrometry, Transfection, Expressing, Plasmid Preparation, One-tailed Test, Western Blot

    a) Up, Representation of the methyl binding domain (MBD)-mediated targeting approach of proteins to methylated CpG. Here, the CXXC mutated KDM2B is redirected to methylated CpG via the MBD. Bottom, Schematic representing the MBD (black square) fusion constructs for Luciferase control (Luc) and KDM2B. The KDM2B long isoform contains the histone demethylase JmjC domain (gold box) and a mutated CXXC domain (dark orange). Both constructs contain a V5 tag. b) Left, Immunofluorescence of human synovial sarcoma (HS-SY-II) cells displaying the MBD constructs (V5, magenta), BCOR (green) and H2AK119ub1 (cyan). Yellow arrow heads point to the MBD foci. Scale bars represents 5μm throughout the figure. Right, quantification of the percentage of BCOR or H2AK119ub1 foci overlapping a V5 foci in 3 or 4 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Left, Immunofluorescence for V5 (magenta) and SS18-SSX1 (HA, cyan). Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 5 biological replicates. d) Left, Immunofluorescence for V5 (magenta) and H3K27me3 (cyan). Right, quantification of the percentage of BCOR or H3K27me3 foci overlapping with V5 foci in 2 biological replicates. Data represents the mean and S.E.M. e) Left, Immunofluorescence of MBD-KDM2B (V5, magenta) in the presence of different sgRNAs (eGFP background fluorescence) with SS18-SSX1 (HA, cyan) in in HS-SY-II-Cas9 cells. Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 4 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (** p< 0.01 and *** p< 0.001). f) Left, Immunofluorescence images of MBD-KDM2B (V5, magenta) with SS18-SSX or H2AK119ub1 or MacroH2A1.1 (cyan) in HS-SY-II-Cas9 cells expressing sgCtrl, sgMacroH2A (targeting both histone genes H2AFY and H2AFY2) or sgPCGFI. Right, quantification of the percentage of foci overlapping MBD-KDM2B foci in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (* p< 0.05 and ** p< 0.01).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Up, Representation of the methyl binding domain (MBD)-mediated targeting approach of proteins to methylated CpG. Here, the CXXC mutated KDM2B is redirected to methylated CpG via the MBD. Bottom, Schematic representing the MBD (black square) fusion constructs for Luciferase control (Luc) and KDM2B. The KDM2B long isoform contains the histone demethylase JmjC domain (gold box) and a mutated CXXC domain (dark orange). Both constructs contain a V5 tag. b) Left, Immunofluorescence of human synovial sarcoma (HS-SY-II) cells displaying the MBD constructs (V5, magenta), BCOR (green) and H2AK119ub1 (cyan). Yellow arrow heads point to the MBD foci. Scale bars represents 5μm throughout the figure. Right, quantification of the percentage of BCOR or H2AK119ub1 foci overlapping a V5 foci in 3 or 4 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). c) Left, Immunofluorescence for V5 (magenta) and SS18-SSX1 (HA, cyan). Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 5 biological replicates. d) Left, Immunofluorescence for V5 (magenta) and H3K27me3 (cyan). Right, quantification of the percentage of BCOR or H3K27me3 foci overlapping with V5 foci in 2 biological replicates. Data represents the mean and S.E.M. e) Left, Immunofluorescence of MBD-KDM2B (V5, magenta) in the presence of different sgRNAs (eGFP background fluorescence) with SS18-SSX1 (HA, cyan) in in HS-SY-II-Cas9 cells. Right, quantification of the percentage of HA (SS18-SSX1) foci overlapping a V5 foci in 2 to 4 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (** p< 0.01 and *** p< 0.001). f) Left, Immunofluorescence images of MBD-KDM2B (V5, magenta) with SS18-SSX or H2AK119ub1 or MacroH2A1.1 (cyan) in HS-SY-II-Cas9 cells expressing sgCtrl, sgMacroH2A (targeting both histone genes H2AFY and H2AFY2) or sgPCGFI. Right, quantification of the percentage of foci overlapping MBD-KDM2B foci in 2 biological replicates. Data represents the mean and S.E.M. Asterisks represent p-values of unpaired one-tailed t-test between groups (* p< 0.05 and ** p< 0.01).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Binding Assay, Methylation, Construct, Luciferase, Immunofluorescence, Fluorescence, One-tailed Test, Expressing

    a) Heatmaps of H2AK119ub1 (purple) or SS18-SSX (blue) CUT&RUN signals in HS-SY-II (left) and SYO-I (right) Cas9 cells expressing empty sgRNA as control (EV) or targeting PCGF1 (sgPCGFI). Both heatmaps represent CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or SS18-SSX2 peaks in SYO-I (right, n=61940). Rows correspond to ±5-kb regions across the midpoint of each enriched region, ranked by increasing signal. b) H2AK119ub1 and SS18-SSX CUT&RUN score distributions across HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or the SS18-SSX2 peaks in SYO-I (right, n=61940). c) Gene track for H2AK119ub1 and SS18-SSX CUT&RUN at the EN2-SHH-NOM1 and FGF4-FGF3 loci. d) Salt extraction assay displaying SS18-SSX1 levels by western blot in HS-SY-II-Cas9 cells expressing an empty vector (EV) or sgRNAs against PCGF1 or PCGF3. e) Quantification of the SS18-SSX protein distribution in the various salt extraction fractions. Data represents the mean and S.E.M for the percentage of total protein per fraction in 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Heatmaps of H2AK119ub1 (purple) or SS18-SSX (blue) CUT&RUN signals in HS-SY-II (left) and SYO-I (right) Cas9 cells expressing empty sgRNA as control (EV) or targeting PCGF1 (sgPCGFI). Both heatmaps represent CUT&RUN signals over HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or SS18-SSX2 peaks in SYO-I (right, n=61940). Rows correspond to ±5-kb regions across the midpoint of each enriched region, ranked by increasing signal. b) H2AK119ub1 and SS18-SSX CUT&RUN score distributions across HA-SS18-SSX1 peaks in HS-SY-II (left, n=52027) or the SS18-SSX2 peaks in SYO-I (right, n=61940). c) Gene track for H2AK119ub1 and SS18-SSX CUT&RUN at the EN2-SHH-NOM1 and FGF4-FGF3 loci. d) Salt extraction assay displaying SS18-SSX1 levels by western blot in HS-SY-II-Cas9 cells expressing an empty vector (EV) or sgRNAs against PCGF1 or PCGF3. e) Quantification of the SS18-SSX protein distribution in the various salt extraction fractions. Data represents the mean and S.E.M for the percentage of total protein per fraction in 3 biological replicates. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Expressing, Western Blot, Plasmid Preparation, One-tailed Test

    a) Log2 Fold change of RPKM values from RNA sequencing in CRL7250 cells expressing SS18 or SS18-SSX1 compared to naive cells in two biological replicates. Data from (McBride et al., 2018). b) Log2 Fold change of RPKM values from RNA sequencing in HS-SY-II and SYO-I cells after knockdown of SS18-SSX compared to shCtrl cells in two biological replicates. Data from (McBride et al., 2018). c, d) Western blot of whole cell extracts from mesenchymal stem cells expressing eGFP or eGFP-SS18-SSX1 (c); and from HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI) (d). Proteins were detected using BCOR and PCGF1 antibodies and Beta-actin was used as loading control. e) Gene tracks for HA-SS18-SSX1 and SS18-SSX2 CUT&RUN at the BCOR and RYBP loci. f) Left, Immunofluorescence of eGFP-fused constructs (eGFP, eGFP-SS18-SSX1) expressed in HS-SY-II with eGFP signals (green) and nucleus stained with DAPI and H2AK119ub1. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). g) Left, H2AK119ub1 immunofluorescence in HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI). sgRNA expressing cells are positive for eGFP. Cells were mixed with non sgRNA expressing cells for direct comparison of H2AK119ub1 signal intensity (yellow arrow heads). Right, quantification of the H2AK119ub1 fluorescence ratio in eGFP (=sgRNA) versus no eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars represents 10μm. Scale bars represents 20μm.

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Log2 Fold change of RPKM values from RNA sequencing in CRL7250 cells expressing SS18 or SS18-SSX1 compared to naive cells in two biological replicates. Data from (McBride et al., 2018). b) Log2 Fold change of RPKM values from RNA sequencing in HS-SY-II and SYO-I cells after knockdown of SS18-SSX compared to shCtrl cells in two biological replicates. Data from (McBride et al., 2018). c, d) Western blot of whole cell extracts from mesenchymal stem cells expressing eGFP or eGFP-SS18-SSX1 (c); and from HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI) (d). Proteins were detected using BCOR and PCGF1 antibodies and Beta-actin was used as loading control. e) Gene tracks for HA-SS18-SSX1 and SS18-SSX2 CUT&RUN at the BCOR and RYBP loci. f) Left, Immunofluorescence of eGFP-fused constructs (eGFP, eGFP-SS18-SSX1) expressed in HS-SY-II with eGFP signals (green) and nucleus stained with DAPI and H2AK119ub1. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). g) Left, H2AK119ub1 immunofluorescence in HS-SY-II-Cas9 cells expressing empty sgRNA vector (EV) or sgRNA against SSX or PCGF1 (sgSSX, sgPCGFI). sgRNA expressing cells are positive for eGFP. Cells were mixed with non sgRNA expressing cells for direct comparison of H2AK119ub1 signal intensity (yellow arrow heads). Right, quantification of the H2AK119ub1 fluorescence ratio in eGFP (=sgRNA) versus no eGFP cells in 3 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars represents 10μm. Scale bars represents 20μm.

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: RNA Sequencing Assay, Expressing, Western Blot, Plasmid Preparation, Immunofluorescence, Construct, Staining, Fluorescence, One-tailed Test

    a) Left, Immunofluorescence against BCOR in HS-SY-II synovial sarcoma cells expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ). Right, quantification of BCOR fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. b) Sequential chromatin washes assay using 150mM salt buffer in untransduced control (Ctrl) or eGFP-SSX-C expressing HEK293T cells. BCOR, PCGF1 or Beta-Actin as a loading control were detected by western blot. c) Quantification of the protein distribution for BCOR, PCGF1 or Beta-Actin in the various washes. Data represents the percentage of total protein levels. d) Left, Immunofluorescence against H2AK119ub1 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 7 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (*** p< 0.001). Scale bars represents 20μm. e) Left, Immunofluorescence against SS18 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of the SS18 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. f) H&E and immunohistochemical staining for Inhibin-a, SSX and H2AK119ub1 in human testis. Scale bar correspond to 40μm in the upper panel. Lower panel is a close-up from the images shown in the upper panel (area marked by a dashed line) where the scale bar corresponds to 20μm.

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Left, Immunofluorescence against BCOR in HS-SY-II synovial sarcoma cells expressing eGFP-fused constructs (eGFP, eGFP-SSX-C and eGFP-SSX-C ΔRD ). Right, quantification of BCOR fluorescence ratio in high versus low eGFP cells in 5 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. b) Sequential chromatin washes assay using 150mM salt buffer in untransduced control (Ctrl) or eGFP-SSX-C expressing HEK293T cells. BCOR, PCGF1 or Beta-Actin as a loading control were detected by western blot. c) Quantification of the protein distribution for BCOR, PCGF1 or Beta-Actin in the various washes. Data represents the percentage of total protein levels. d) Left, Immunofluorescence against H2AK119ub1 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of H2AK119ub1 fluorescence ratio in high versus low eGFP cells in 7 biological replicates. Data represents the mean and the S.E.M. Asterisks represent p-values of paired one-tailed t-test between groups (*** p< 0.001). Scale bars represents 20μm. e) Left, Immunofluorescence against SS18 in HS-SY-II cells expressing the indicated eGFP-fused constructs. Right, quantification of the SS18 fluorescence ratio in high versus low eGFP cells in 2 biological replicates. Data represents the mean and the standard error of the mean (S.E.M). Asterisks represent p-values of paired one-tailed t-test between groups (* p< 0.05). Scale bars correspond to 20μm. f) H&E and immunohistochemical staining for Inhibin-a, SSX and H2AK119ub1 in human testis. Scale bar correspond to 40μm in the upper panel. Lower panel is a close-up from the images shown in the upper panel (area marked by a dashed line) where the scale bar corresponds to 20μm.

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Immunofluorescence, Expressing, Construct, Fluorescence, One-tailed Test, Western Blot, Immunohistochemical staining, Staining

    a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Overview of the Hic1 CreERT2 knock-in allele (Scott et al, 2019) and of the Rosa26 hSS2 (also known as SSM2) allele (Haidar et al, 2007) for conditional induction of SS18-SSX2 in Hic1-expressing mesenchymal progenitors. Upon tamoxifen treatment CreERT2 mediates recombination between the two LoxP sites in SSM2 mice, thereby removing the transcriptional stop signal and allowing transcription of SS18-SSX2-IRES-EGFP from the endogenous ROSA26 promoter. b) Illustration of the time line for the tissue sample collection of samples analysed in (c, d) 8-week-old mice were treated with tamoxifen and tongue muscle tissues were collected at 5, 7 and 9 weeks post-induction. and were created with BioRender.com. c) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 , mice tongue tissue at 5, 7 or 9 weeks after induction by tamoxifen treatment. The cells are stained for DAPI, SSM2 (eGFP) and H2AK119ub1. The scale bar represents 100 μm. d) Quantification of H2AK119ub1 signal intensity in normalised to DAPI signal intensity in 5 or 4 biological replicates of mice treated with tamoxifen (TAM) in SSM2 (GFP) negative tongue muscle or in adjacent SSM2 positive cell clusters. Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01). e) limmunohistochemical staining for H2AK119ub1 on a tissue microarray of human surgical excised tissue specimens (left: skeletal muscle; right: synovial sarcoma). Scale bars correspond to 50μm in the left panel. f) Quantification of H2AK119ub DAB signal intensity across 37 synovial sarcomas (sample cores in duplicate), other sarcomas (1 case each of epithelioid sarcoma, sarcomatoid mesothelioma, Ewing sarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma, dedifferentiated liposarcoma, and myxoid liposarcoma) and normal tissues (normal skeletal muscle, ovarian stroma, breast glandular tissue, and testis controls). Quantification for the two skeletal muscle samples is also shown separately in the graph. All samples were stained in parallel on the same formalin-fixed, paraffin embedded tissue microarray slide. Asterisks represent p-values of Mann-Whitney test between groups (** p< 0.01).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Knock-In, Expressing, Immunofluorescence, Staining, One-tailed Test, Microarray, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: a) Immunofluorescence of Hic1 creERT2/creERT2 ; Rosa26 SSM2/SSM2 mice at 16-week endpoint tongue tissue showing left, samples from control mice not treated with tamoxifen (-TAM) (upper panel) or from tamoxifen treated (+TAM) mice expressing the SSM2 cassette (human SS18-SSX2) embedded in striated muscle (lower panel). The cells are stained for DAPI, SSM2 and H2AK119ub1. The scale bar represents 100 μm. b) Close-ups of images shown in the panel above, area corresponds to dashed line in (a). c) Quantification of H2AK119ub signal intensity normalised to DAPI signal intensity in 3 biological replicates in -TAM control mice, or +TAM tongue muscle or adjacent tumours (SSM2 negative or positive respectively). Asterisks represent p-values of paired one-tailed t-test between groups (** p< 0.01).

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: Immunofluorescence, Expressing, Staining, One-tailed Test

    Model depicting the strong interplay between SS18-SSX and H2AK119ub1 where SS18-SSX interacts with histones H2A which are ubiquitinated on their lysine K119. SS18-SSX then promotes further levels of H2AK119ub1 via two distinct mechanisms: 1) by stimulating the transcription of PRC1.1 members BCOR and RYBP as direct targets of the fusion and 2) by increasing the stability of the PRC1.1 complex on chromatin. In both cases H2AK119ub1 levels increase and therefore reinforce SS18-SSX’s presence on chromatin.

    Journal: bioRxiv

    Article Title: An autoregulatory feedback loop converging on H2A ubiquitination drives synovial sarcoma

    doi: 10.1101/2022.07.18.499263

    Figure Lengend Snippet: Model depicting the strong interplay between SS18-SSX and H2AK119ub1 where SS18-SSX interacts with histones H2A which are ubiquitinated on their lysine K119. SS18-SSX then promotes further levels of H2AK119ub1 via two distinct mechanisms: 1) by stimulating the transcription of PRC1.1 members BCOR and RYBP as direct targets of the fusion and 2) by increasing the stability of the PRC1.1 complex on chromatin. In both cases H2AK119ub1 levels increase and therefore reinforce SS18-SSX’s presence on chromatin.

    Article Snippet: The primary antibodies H2AK119Ub (Cell Signaling Technology, #8240, Danvers, MA, USA), and SSX-SS18 (Cell Signaling Technology, #72364S, Danvers, MA, USA) were incubated at 1:400 for 30 min and 1:300 for 15 min, respectively, at ambient temperature.

    Techniques: