Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) UMAP plot of integrated single-cell RNA sequencing (scRNAseq) datasets of mouse embryonic developing colon (hindgut), healthy adult colon and colon cancer (derived from AKPS organoid-based model). (B) Volcano plots of dual differential gene expression analysis comparing epithelial cell gene expression from scRNAseq data of embryonic hindgut and colon cancer to the healthy adult colon. Highlighted genes are examples of common genes in the embryonic and cancer cells. (log2FC (log2 fold change) > 0.6, p_adj.glb (p value adjusted global) < 0.05). Differential expression and p values were calculated via hierarchical permutation tests. (C) Immunofluorescent (IF) staining on embryonic hindgut, healthy adult colon and colon cancer for Tactsd2 and Marcksl1 (magenta). Blue DAPI. Scale bar: 50 μm. (D) Regulatory network inference using SCENIC analysis on the integrated epithelial scRNAseq data (from 1A). (E) Expression values of SoxC genes are shown as psupertime value learned for each cell (X-axis) and Z-scored log2 gene expression values (Y-axis) across three embryonic and one adult time-points. (F) Kaplan Meier plots showing the inverse correlation between SOXC family gene expression and CRC patients’ relapse-free survival. Hazard ratio (HR) and logarithmic ranked p Value (logrank P) were analyzed to infer the significance of the differences. Numbers below each graph represent number of patients at risk in any given time (months), black for low expression and red for high expression of each gene. The cut-off is automatically calculated based on the best performing threshold. (G) IF staining on embryonic hindgut, healthy adult colon and colon cancer for Sox11 (grey). Blue DAPI. Scale bar: 50 μm. (H) IF staining on human healthy and malignant colon for EPCAM (epithelial marker, green) and SOX11 protein (magenta). Blue DAPI. Scale bar: 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Staining, Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Predicted functional gene interaction network of oncoembryonic genes derived from dual differential gene expression analysis (from ), showing significant interactions among key SoxC-regulated genes. (B) Summary of gene ontology (GO) terms represented in panel S3A, displayed as a pie chart, with numbers indicating the percentage of GO terms in each category. (C) Complete regulatory network inference using SCENIC analysis on integrated epithelial scRNAseq data (from ), highlighting Sox4 and Sox11 as master regulators in the oncoembryonic cells. (D) Quantitative PCR showing transcript levels of SoxC transcription factor targets in healthy, healthy-adjacent, and colon tumor tissues from mouse models. (E) RNA in situ hybridization (RNAscope) and quantification of signal intensity for Sox4 and Sox11 in mouse healthy and colon tumor tissues. Scale bar: 50 μm, DAPI (blue) counterstains nuclei. (F) Immunofluorescent staining of human healthy and cancerous colon tissues for E-CADHERIN (epithelial marker) and SOX11 protein. Scale bar: 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Functional Assay, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, RNA In Situ Hybridization, RNAscope, Staining, Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Ridge plot representing normalized counts of Sox11, Sox4 and Sox12 in epithelial cells at different stages of colon development. (B) Full R26-CreERT2; SoxC fl/fl or non-Cre littermate embryos at stage E14.5 or E18.5 after tamoxifen induction of Cre at E10.5. Arrows point at eye, back and digit phenotypes. (C) Length (in mm) of SoxC-KO or non-Cre littermate midguts at E18.5. (D) Quantification of mesenchymal nuclei present in the region below the epithelium and before the smooth muscle layer in SoxC-KO or non-Cre littermate E14.5 hindguts. (E) Marker genes were used for the annotation of the integrated WT and SoxC-KO E14.5 hindgut scRNAseq dataset. (F-H) Gene ontology and network analyses of top 200 genes enriched in clusters Epithelial 3 (F), Epithelial 4 (G) and Mesenchymal 4 (H) of the integrated WT and SoxC-KO E14.5 hindgut scRNAseq dataset.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Length of entire hindguts in SoxC-KO E14.5 embryos and non-Cre (unrecombined SoxC fl/fl) littermates. (B) Representative immunofluorescence (IF) staining of SoxC-KO and non-Cre littermates E14.5 hindguts (blue DAPI, green Phalloidin, grey E-Cadherin). Scale bar: 50 μm. (C) Measurement of stromal widths (defined as the distance between the lower epithelial border and the beginning of the smooth muscle layer) in multiple images similar to 2B. Each dot represents one measurement, between 3 to 5 measurements were taken from n=3 SoxC-KO and n=3 WT hindguts. (D) Schematics of the multi-omics experiments performed using E14.5 wild-type (WT) and SoxC-KO hindguts. (E) UMAP plot of SoxC-KO E14.5 hindgut scRNAseq integrated with previously published E14.5 WT hindgut scRNAseq. Dots represent single cells, colored by cell type as illustrated in . (F) Volcano plot of gene expression fold changes in scRNAseq of E14.5 hindguts in SoxC-KO or WT mice. Magenta dots represent genes significantly downregulated in SoxC-KO (log2FC (log2-fold change) < -0.6, p_adj.glb (p value adjusted global) < 0.05), blue dots represent genes significantly upregulated in SoxC-KO (log2FC > 0.6, p_adj.glb < 0.05). Selected genes are labeled. Differential expression and p values were calculated via hierarchical permutation tests. (G) Volcano plot of ATACseq peaks in SoxC-KO and WT E14.5 hindguts as identified via the ATACseq snakePipes pipeline. Magenta dots represent peaks significantly lost or reduced in SoxC-KO compared to WT (logFC < -1, FDR < 0.05), blue dots represent peaks significantly gained or increased in SoxC-KO compared to WT (logFC > 1, FDR < 0.05). (H) ChIPseq profiles in selected genomic regions bound by Sox4 and Sox11, showing two examples of positively regulated (top) and two negatively regulated (bottom) genes, also identified in scRNAseq and ATACseq (2F&G). (I) Number of genes with significant changes detected in scRNAseq, ATACseq or ChIPseq and schematics of overlaps between the three assays. Note that the Sankey plot shows only the genes shared between the datasets, while the total number of genes in each category exceeds the number of shared genes. (J) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, grey Sox11, magenta Klf4, green Phalloidin) and quantification of nuclear Klf4 measured as averaged intensity of all nuclei in the section. (K) qPCR analysis of Klf4 expression in WT and SoxC-KO E14.5 hindguts. (L) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, grey E-Cadherin, magenta Tead2, green Phalloidin) and quantification of nuclear Tead2 measured as averaged intensity of all nuclei in the section. (M) qPCR analysis of Tead2 expression in WT and SoxC-KO E14.5 hindguts. (N) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, magenta Mdk, green Phalloidin) and quantification of nuclear Mdk measured as averaged intensity of all nuclei in the section. (O) qPCR analysis of Mdk expression in WT and SoxC-KO E14.5 hindguts. Statistical analyses for 2B and 2J-O were performed using a two-tailed unpaired t-test. Scale bars 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Immunofluorescence, Staining, Expressing, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A-C) Gene ontology analysis (biological processes) of all genes nearest to genomic regions with lost accessibility (A) or gained accessibility (B) in SoxC-KO E14.5 hindgut compared to WT or with significant ChIPseq peaks for Sox11 and or Sox4 (C). Top 20 gene ontologies are shown. (D) Representative immunofluorescent staining in SoxC-KO or non-Cre littermate E14.5 hindguts. Blue DAPI, magenta Klf5, green Phalloidin. Scalebar 50 μm. (E) qPCR analysis of Klf5 expression in WT and SoxC-KO E14.5 hindguts. Statistical analysis was performed using a two-tailed unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Schematics of the generation of AKPS SoxC-KO organoids. (B) Primary tumor morphology of control (Cas12a intergenic targeting) and SoxC-KO (Cas12a targeting Sox4, Sox11 and Sox12 each with two specific gRNAs). Scalebar: 5 mm. (C) Bar plot of the tumor weights in the two groups. (D) Percentage of primary tumor and metastatic growth. (E) Quantitative PCR showing the transcript levels of SoxC TFs targets, Mdk and Tead2 . (F) Immunofluorescent staining of Mdk in Control and SoxC-KO colorectal cancer (CRC) tumors. Scalebars: 50 μm. Statistical analyses were performed using an unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Control, Real-time Polymerase Chain Reaction, Staining

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Immunofluorescent (IF) staining of Mdk (magenta) in E14.5 hindgut, healthy adult colon and colon cancer. Blue DAPI. (B) IF staining of Tead2 (magenta) in murine E14.5 hindgut, healthy adult colon and colon cancer. Blue DAPI. (C) Survival analysis of colorectal patients divided into high or low MDK expression. (D) Inferred interactions through Mdk signaling in E14.5 WT hindgut. Edge weights are proportional to the interaction strength. (E) IF staining and quantification of epithelial thickness of E13.5 hindgut explants cultured ex-vivo for 2 days in the presence of MDK inhibitor (iMDK, n=6) or control (DMSO, n=6). For each explant, 5-7 measurements were taken. Blue DAPI, grey E-cadherin, green Ki67. (F) Survival analysis of colorectal patients divided into high or low TEAD2 expression. (G) IF staining and quantification of epithelial thickness of E12.5 hindgut explants cultured ex-vivo for 2 days in the presence of TEAD inhibitor (iTEAD, n=4) or control (DMSO, n=3). For each explant, 5-7 measurements were taken. Blue DAPI, grey E-cadherin, green Ki67. (H) Activity of Tead2 promoter after Sox11 or Sox4 transfection assessed by luciferase assay in HEK293 cells. N=8 for Sox11, n=3 for Sox4. (I) IF staining of Klf4 and Sox11 in mouse colon cancer and adjacent tissue. Insets of representative regions with high nuclear Klf4 or Sox11 expression. On top: quantification of mean nuclear intensity along a left-to-right axis. Scale bars: 50 μm. Statistical analyses for (D), (G), (H) using two-tailed unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Staining, Expressing, Cell Culture, Ex Vivo, Control, Activity Assay, Transfection, Luciferase, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Comparison of inferred information flow for all predicted cell-communication pathways in E14.5 hindgut or adult colon. Red-colored pathways are enriched in the E14.5 hindgut, blue-colored pathways are enriched in the adult. (B) Significant predicted Mdk ligand-receptor interactions from cell-cell communication prediction by CellChat in the scRNAseq of E14.5 WT including all cell types. Edge weights are proportional to the interaction strength. (C) Significant predicted Mdk ligand-receptor interactions originating from epithelial cells (Oncoembryonic/ISC-like, Enterocyte-like, Tuft-like, Secretory-like or Enteroendocrine-like). Inferred interactions are the result of cell-cell communication prediction by CellChat in the scRNAseq of cancer cells only (AKPS organoid-derived tumors) including all cell types. Edge weights are proportional to the interaction strength. (D) Inferred interactions through Mdk signaling from epithelial cells as in (B). (E) Quantification of nuclear Ki67 signal in and replicates (E13.5 hindgut explants cultured for 2 days ex vivo in the presence of DMSO or MDK inhibitor). (F) Quantification of nuclear Ki67 signal in and replicates (E12.5 hindgut explants cultured for 2 days ex vivo in the presence of DMSO or TEAD inhibitor). (G) Top: Activity of WT Tead2 promoter or with deletion of putative Sox11 binding site (BS) after Sox11 transfection assessed by luciferase assay in HEK293 cells. N=8 for WT, n=3 for BS mutation. Bottom: schematics of Tead2 promoter region cloned for the assay (−465 to +94 from the coding sequence start) and the mutated base (position +10 from the coding sequence start). Statistical analysis using two-way Anova with Bonferroni’s multiple comparison test. (H) Schematics of strategy to identify intermediate regulators of indirect targets of SoxC TFs. (I) Representative immunofluorescent staining of adult mouse colon (left, blue DAPI, green Pdgfra-GFP, magenta Klf4) or human healthy colon (right, blue DAPI, green E-CADHERIN, magenta KLF4). (J) Z-scored log2 expression values of Klf3, Klf4, Klf5, Klf6 with cell ordered along a pseudotime learned by pstupertime at different stages of colon development (x-axis). (K) Immunofluorescent staining from (mouse colon cancer) with nuclear mask applied on each separate channel. Top: grey, Klf4. Middle: magenta, Sox11. Bottom: blue DAPI, grey Klf4, magenta Sox11. If not otherwise stated, statistical analyses were performed using a two-tailed unpaired t-test. Scale bars 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Comparison, Derivative Assay, Cell Culture, Ex Vivo, Activity Assay, Binding Assay, Transfection, Luciferase, Mutagenesis, Clone Assay, Sequencing, Staining, Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: Expression of SOX4 (A), SOX12 (B) and SOX11 (C) in the MCL cell lines Granta 519, JeKo, Rec1 and JVM2 by quantitative RT- PCR. Granta 519 express high levels of SOX11 mRNA but low levels of the other SOXC genes while no SOX11 mRNA expression was detected in JVM2. (D) WB showed that Granta 519, JeKo and Rec1, but not JVM2, express SOX11 protein.

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: (A) Expression of SOX11 was evaluated after transfection with either 100 pmol SOX11 siRNA or control siRNA. The SOX11 mRNA level was measured by quantitative RT-PCR 20 hours after electroporation. Error bars show standard deviation of 4 independent experiments, ***p<0.001. (B) Western blots show downregulation of SOX11 protein in Granta 519 after SOX11 siRNA knockdown but not in cells treated with control siRNA or in untransfected cells. (C) Depletion of SOX11 protein significantly reduces TUBB3 expression. Granta 519 cells were transfected with SOX11 siRNA and TUBB3 expression was investigated by quantitative RT-PCR after 20 hours. ***P<0.001.

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Electroporation, Standard Deviation, Western Blot, Knockdown

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: Differently expressed genes after SOX11 knockdown by siRNA in Granta 519 cells # .

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Knockdown, Sequencing

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: Validation of SOX11 siRNA downregulated genes in a dataset of 16 primary MCL a) .

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Biomarker Discovery

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: Validation of SOX11 siRNA downregulated genes in a series of SOX11+ and SOX11- MCL a) .

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Biomarker Discovery

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: Analysis by qRT-PCR confirms downregulation of DBN1 and SETMAR (A) in SOX11 siRNA treated Granta 519 cells. (B) Expression of SOX11, TUBB3, SETMAR and DBN1 in the JeKo cell line was evaluated after transfection with either 100 pmol SOX11 siRNA or control siRNA. mRNA levels were measured by quantitative RT-PCR 20 hours after electroporation. Error bars show standard deviation of 4 independent experiments. * P<0.05, **P<0.01.

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Electroporation, Standard Deviation

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: The expression values of SOX11, DBN1 or SETMAR were analyzed by quantitative RT-PCR. The threshold cycle (Ct) of the gene against the Ct of β actin was determined by subtraction. All values were first tested for normal distribution, and then the Pearson correlation coefficients and P value were calculated. ▴ MCL cell lines Granta 519, Rec1 and JeKo; • primary MCL patients.

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Gene Expression Profiling and Chromatin Immunoprecipitation Identify DBN1, SETMAR and HIG2 as Direct Targets of SOX11 in Mantle Cell Lymphoma

doi: 10.1371/journal.pone.0014085

Figure Lengend Snippet: ChIP assay of SOX11 protein binding to the selected three genes. Immunoprecipitated DNA was analyzed by quantitative RT-PCR for two different primers as described in material and method. TSS: primer designed close to transcription start site (TSS) and DT: primer distal (∼2 kb) to the TSS. Columns represent the mean of fold enrichment of DBN1, SETMAR or HIG2 relative to IgG control. Error bars show standard deviation of 3 independent experiments.* P<0.05, ** P<0.01 compared to the IgG control.

Article Snippet: The blotted membranes were probed with rabbit anti-human SOX11 (HPA000536, Atlas Antibodies, Uppsala, Sweden), and antibodies to tubulin (SC 8035, Santa Cruz Biotechnology, Santa Cruz, CA) or actin (A4700, Sigma, Saint Louis, MO) was used as loading controls.

Techniques: Protein Binding, Immunoprecipitation, Quantitative RT-PCR, Control, Standard Deviation

Journal: Science advances

Article Title: Usp11 controls cortical neurogenesis and neuronal migration through Sox11 stabilization.

doi: 10.1126/sciadv.abc6093

Figure Lengend Snippet: Fig. 5. Usp11 deubiquitinates Sox11. (A) The volcano plot of the proteins identified from WT and Usp11 KO cortices by label-free quantitative LC-MS/MS. The proteins are plotted according to their log2 fold changes (x axis) and log10 P values (y axis). Proteins down-regulated in Usp11 KO are shown as black dots. Among them, proteins with known functions in neurogenesis or nervous system development are marked in red, the known targets of Sox11 are in blue, and the protein with both features is in orange. (B) Venn diagram of the number of proteins down-regulated in Usp11 KO cortices uncovered from proteome analysis (four biological replicates) overlapping with that showing a decreased ubiquitination in Usp11 overexpressed neurons identified from ubiquitylome analysis (one biological replicate). (C and D) Analysis of Sox11 ubiquitination levels in 293T cells transfected with indicated constructs (C) or N2a cells stably expressing indicated shRNAs and transfected with indicated constructs (D). The cellular ubiquitinated proteins were pulled down by Ni-NTA beads under denaturing conditions and then analyzed by Western blot. (E) In vitro deubiquitination assay using purified and ubiquitinated Sox11 and separately purified Usp11. (F and G) Immunoprecipitation analysis of the interaction between endogenous Usp11 and en- dogenous Sox11 in N2a cells (F) or lysate of mouse cortex (G). IgG, immunoglobulin G.

Article Snippet: Hes1 and Sox11 cDNAs were purchased from Origene and subcloned to pRK5-Flag and/or pUS2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Transfection, Construct, Stable Transfection, Expressing, Western Blot, In Vitro, Purification, Immunoprecipitation

Journal: Science advances

Article Title: Usp11 controls cortical neurogenesis and neuronal migration through Sox11 stabilization.

doi: 10.1126/sciadv.abc6093

Figure Lengend Snippet: Fig. 6. USP11 stabilizes Sox11 to result in a robust Sox11 induction during cortical development. (A and B) Western blot analysis of Sox11 expression in N2a cells expressing indicated shRNAs (A) or in 293T cells transfected with indicated constructs (B). (C and D) Western blot analysis of Sox11 expression in N2a cells stably expressing indicated shRNAs and treated with MG132 for 16 hours (C) or cycloheximide for indicated time points (D). Of note, for an accurate comparison of protein turn- over, a higher amount (2.5-fold) of lysates from Usp11 knockdown cells was loaded (D). The levels of Sox11 are normalized to that in 0 hours and indicated on the bottom. (E and F) RT–quantitative PCR (qPCR) (E) and Western blot (F) analyses of the expression of indicated genes/proteins at indicated time points during neuron induction using NPCs isolated from E12.5 cerebral cortices. The relative amounts of Sox11 are indicated on the bottom (F). (G) RT-qPCR analysis of the expression of Sox11 mRNA in the cortices of Usp11 KO or wild-type littermates at indicated devel- opmental stages. (H and I) Western blot analysis of Sox11 expression in the cortices of Usp11 KO or wild-type littermates at indicated developmental stages. The three repeats of Western blots and quantitative data are shown in (H) and (I), respectively. In (E), (G), and (H), data are means ± SD, n = 3.

Article Snippet: Hes1 and Sox11 cDNAs were purchased from Origene and subcloned to pRK5-Flag and/or pUS2.

Techniques: Western Blot, Expressing, Transfection, Construct, Stable Transfection, Comparison, Knockdown, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

Journal: Science advances

Article Title: Usp11 controls cortical neurogenesis and neuronal migration through Sox11 stabilization.

doi: 10.1126/sciadv.abc6093

Figure Lengend Snippet: Fig. 8. Disease-associated Usp11 mutant is defective in regulating Sox11. (A) Western blot analysis of SOX11 expression in 293T cells expressing Usp11 WT or R241Q mutant. (B) Analysis of Sox11 ubiquitination levels in 293T cells transfected with indicated constructs. Sox11 was immunoprecipitated from cell lysates and analyzed by Western blot. (C) Immunoprecipitation analysis of the interaction of wild-type or mutant Usp11 with endogenous SOX11 in 293T cells. To prevent SOX11 degradation, the transfected cells were treated with MG132 for 16 hours before harvest. (D and E) Immunostaining of Sox11 using cortical sections of E15.5 embryos electroporated in utero with indicated shRNA and cDNA constructs and mCherry expressing construct at E12.5. The transfected cells are visualized by mCherry and marked by arrows. Scale bar, 20 m. The Sox11 immunofluorescence intensities in mCherry+ cells were quantified and shown in (E). Data are means ± SD; **P < 0.01 and ***P < 0.001 by t test, n = 45.

Article Snippet: Hes1 and Sox11 cDNAs were purchased from Origene and subcloned to pRK5-Flag and/or pUS2.

Techniques: Mutagenesis, Western Blot, Expressing, Ubiquitin Proteomics, Transfection, Construct, Immunoprecipitation, Immunostaining, In Utero, shRNA, Immunofluorescence

Journal: Science advances

Article Title: Usp11 controls cortical neurogenesis and neuronal migration through Sox11 stabilization.

doi: 10.1126/sciadv.abc6093

Figure Lengend Snippet: Fig. 7. Sox11 stabilization contributes to the functions of Usp11 in cortical de- velopment. (A) Immunostaining for Tbr1 and DAPI staining of cortical sections of E15.5 embryos electroporated in utero with indicated constructs and mCherry ex- pressing construct at E12.5. The transfected cells are marked by mCherry. Scale bar, 100 m. (B and C) Quantification of results from (A) showing the percentage of transfected cells expressing Tbr1 (B) and the location of transfected cells (C). Data are means ± SD; **P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test, n = 4. (D) Immunostaining for Satb2 and DAPI staining of cortical sections of E18.5 embryos electroporated in utero with indicated constructs and mCherry expressing construct at E15.5. The transfected cells are marked by mCherry. Scale bar, 100 m. (E and F) Quantification of results from (D) showing the locations of mCherry+Satb2+ cells (E) and mCherry+ cells (F). Data are means ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test, n = 4.

Article Snippet: Hes1 and Sox11 cDNAs were purchased from Origene and subcloned to pRK5-Flag and/or pUS2.

Techniques: Immunostaining, Staining, In Utero, Construct, Transfection, Expressing

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. Log2 copy number ratio on chromosome 2p (4.4–7.1Mb, hg19) showing shortest region of overlap (SRO) (chr2: 5,828,671-6,098,736; hg19) for high-level focal 2p gains (log2 ratio > 0.5) and amplification (log2 ratio > 2) in 5 NB tumors and 1 NB cell line encompassing the SOX11 locus. Tumor 1 is evaluated by arrayCGH, tumor 5 by whole genome sequencing, and tumor 2, 3, 4 and the cell line by shallow whole genome sequencing. B. FISH analysis shows independent amplification of SOX11 (green) and MYCN (red) in two of the tumor cases with SOX11 amplification from (asterix). C. SOX11 (log2) mRNA levels according to copy number status (amplification (log2ratio>2), gain (log2ratio>0.3) or normal) in a cohort of 276 patients (NRC cohort, GSE85047) (T-test, p=1.82e-09). The two samples with SOX11 amplification are tumor 4 and 5 from . D. Kaplan-Meier analysis (overall survival) of 276 neuroblastoma patients (NRC NB tumor cohort, GSE85047) with high or low SOX11 (log2) expression (using highest quartile as cut-off) (p=4.19e-07). E. Immunohistochemical staining for SOX11 on a tissue Micro-array (TMA) of 56 NB tumors and correlation of SOX11 protein levels (cut-off of H-score 50) with overall worse survival. For each group immunohistochemical staining of one tumor is depicted on the right (p=0.0262). F. SOX11 expression (log2) in NB tumors and NB cell lines (blue) as compared to tumors or cell lines from other entities. Boxplots show the 1st quartile up to the 3rd quartile of the data values and median as a line within the box. Whiskers represent the values of the outer two quartiles maximized at 1.5 times the size of the box. If one or more values outside of the whiskers are present, then this is indicated with a single dot next to the implicated whisker. G. Log2 SOX11 (log2) mRNA expression according to ADRN/MES ratio of the activity score (rankSum) for ADRN and MES signature in NB cell lines based on mRNA expression levels (n=28, p=3.94e-24, r=0.889, pearson).

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Amplification, Sequencing, Expressing, Immunohistochemical staining, Staining, Microarray, Whisker Assay, Activity Assay

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: a. Log2 copy number ratio on chr2p (2-18 Mb, hg19), encompassing SOX11 and MYCN , for the 5 NB tumors and 1 NB cell line depicted in . b. Kaplan-Meier analysis of 276 (GSE85047) and 498 neuroblastoma patients (GSE62564) with high or low SOX11 expression (highest quartile cut-off). c. IHC for SOX11 with two independent SOX11 antibodies on a TMA of NB tumors showing correlation of SOX11 levels (n=71, r=889, p=3.94e-24, Spearman correlation) confirming specificity. d. S OX11 (log2) expression in MYCN amplified high risk tumors as compared to MYCN non-amplified high risk tumors and low risk tumors (GSE62546, n=498). ANOVA test followed by Tukey post-hoc test. e . Copy number ratio (log2) of SOX11 in pan-cancer dataset (CCLE) with high copy number for SOX11 in NB (blue) as compared to other tumor entities (gray). f. Genome-wide methylation profile evaluated by reduced representation bisulfite sequencing (RRBS) of SOX11 in CCLE dataset. SOX11 show a low methylation profile in NB (blue) as compared to other entities (gray). g. SOX11 (log2) expression in neuroblasts compared to cortex tissue in 3 foetal adrenal glands, during induced differentiation of human sympatho-adrenal precursor cells along the sympatho-adrenal lineage and in NB tumors. h. SOX11 (log2) expression in tumors of ALK , LIN28B , LSL-MYCN and TH-MYCN mouse models and mouse adrenal glands. i. SOX11 (log2) mRNA expression in sympathetic ganglia containing hyperplastic lesions and advanced tumors of TH-MYCN +/+ mice during MYCN driven NB tumor formation (lightblue) and in normal sympathetic ganglia (darkblue). Error bars represent standard deviation of 4 biological replicates (moderated t-test of Limma Voom, adjusted P value = 7.63e-6). j. MYCN, SOX11 and Vinculin protein levels in 29 NB cell lines, annotated as MES-type (M), MIXED-type (Mi), ADRN-type (A) or not dermined (nd) according to Van Groningen et al. , Boeva et al. , and the ADRN/MES signature on mRNA expression level . k. Activity score (rankSum) for ADRN and MES signature in 283 NB tumors (NRC NB tumor cohort, GSE85047) based on mRNA expression levels. SOX11 (adrenergic, top) and PRRX1 (mesenchymal, bottom) expression is represented for each tumor by size. Boxplots in Figure D, E and I show the 1st quartile up to the 3rd quartile of the data and median is a line within the box. Whiskers show the outer two quartiles maximized at 1.5 times the size of the box.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Expressing, Amplification, Genome Wide, Methylation, Methylation Sequencing, Standard Deviation, Activity Assay

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. H3K27ac activity in a region downstream of SOX11 (chr2, 5.7–7.0Mb, hg19) in 6 adrenergic (CLB-GA, CLB-MA, NB-69, IMR-32, SJNB6, SKNFI, SKNDZ) and 1 mixed (SK-N-SH) (blue), 2 mesenchymal (SH-EP, GI-ME-N) (orange) and a non-malignant neural crest cell line hNCC and non-embryonal breast cancer cell line MCF-7 (green). Signal represents RPKM normalised ChIP signal, super-enhancers are annotated using LILY (red bar). B. SOX11 mRNA correlation with SILC1 mRNA levels in NB cell lines (n=11, p-value=0.00806, r=0.773, Spearman correlation) using the RNA Atlas (total RNA-sequencing). MYCN status is depicted for each cell line. C. SILC1 (log2) mRNA expression in NB cell lines (blue) as compared to cell lines from other entities (gray). D. 4C-seq analysis of the promoter site and SE region downstream of SOX11 (chr2, 5.6-7.1Mb, hg19) in the NB cell lines CLB-GA (blue) and SH-EP (orange) with inclusion of published and unpublished ChIP tracks for H3K27ac, H3K4me1, PHOX2B, HAND2 and GATA3 in CLB-GA, and ATAC and H3K4me3 in SH-EP. Signal represents log likelihood ratio for the ChIP signal as compared to the input signal (RPM normalised). The viewpoint is located at the SOX11 TSS and SILC1 TSS site (cut out 100 kb). Super-enhancers of CLB-GA are annotated using LILY (blue bar).

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Activity Assay, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: a. H3K27ac activity in a region downstream of SOX11 (chr2, 5.7–7.0Mb, hg19) in 11 adrenergic (NB-EBc1, NGP, CLB-PE, CHP-212, LAN-1, N-206, SH-SY5Y, SJ-NB1, SJ-NB8, SK-N-BE(2c), TR-14) and 1 mixed (SK-N-AS) cell line (blue). Signal represents RPKM normalised ChIP signal, super-enhancers are annotated using LILY (red bar). b. H3K27ac activity in a region downstream of SOX11 (chr2, 5.73-6.28Mb, hg19) in SH-Y5Y cells with or without NOTCH3-IC overexpression, the latter resulting in transition from ADRN-type to MES-type cells. The position of the SOX1 downstream super-enhancer is annotated with a rectangle. c. SOX11 (log2) mRNA expression correlated with SILC1 log2 mRNA levels in primary NB tumors (n=497, p=3.01e-12, r=0.308, Spearman correlation). MYCN status is depicted for each tumor. d. SILC1 (log2) expression in neuroblasts as compared to cortex tissue in 3 fetal adrenal glands, during induced differentiation of human sympatho-adrenal precursor cells along the sympatho-adrenal lineage and in NB tumors e. SOX11 and SILC1 log2 expression in the adrenergic subtypes (blue) derived from the isogenic pairs compared to the mesenchymal subtypes (orange) (n=4 pairs). f. ATAC-seq and H3K27ac ChIP-seq in SHEP-cells for the super-enhancer regulated mesenchymal markers SOX9 and ID1, showing the validity of the SHEP H3K27ac ChIP-seq and ATAC-seq data used in . Signal represents RPKM normalised ChIP signal, super-enhancers are annotated using LILY (red bar). For figure D and E, boxplots are drawn as a box, containing the 1st quartile up to the 3rd quartile of the data values. The median is represented as a line within the box. Whiskers represent the values of the outer two quartiles maximized at 1.5 times the size of the box.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Activity Assay, Over Expression, Expressing, Derivative Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. SOX11 protein levels 6 days upon shSOX11 treatment in NGP, CLB-GA and IMR-32 cells with 4 different shRNAs and one non-targeting control (NTC). Vinculin (VCL) is used as loading control. B. SOX11 protein levels 48h and 72h upon si SOX11 treatment in IMR-32 cells (nucleofection). SOX11 protein levels 24h upon siRNA treatment in NGP, CLB-GA and SK-N-AS cells (dharmafect transfection). Vinculin (VCL) is used as loading control. C. Reduction in colony formation capacity for NGP, CLB-GA and SK-N-AS cells, 14 days upon siRNA SOX11 treatment (dharmafect transfection) as compared to non-targeting control (siNTC). D. Cell cycle analysis 6 days upon shRNA SOX11 treatment in the NGP and CLB-GA cell line. G1 cell cycle arrest upon SOX11 knockdown as compared to the non-targeting control (NTC). Mann-Whitney statistical test is based on G1 phase percentage (shSOX11 vs NTC: NGP p-value=0.2, CLB-GA p-value=0.2, IMR-32 p-value=0.2). Data-points were mean-centered and auto scaled, error bars represent the 95% CI of three biological replicates for every cell line. E. Reduced proliferation (% confluence) over time upon prolonged SOX11 knockdown with 2 different shRNAs for 4 days as compared to non-targeting control (NTC) in NGP and IMR-32 cells. Error bars represent the 95% CI of 3 biological replicates. ANOVA test followed by Tukey post-hoc test (NGP: sh3 vs NTC p=4.51e-14, sh4 vs NTC p=5.06e-14; IMR-32: sh3 vs NTC p = 2.16e-14, sh4 vs NTC p=2.74e-06).

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Control, Transfection, Cell Cycle Assay, shRNA, Knockdown, MANN-WHITNEY

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. Top 50 up- and downregulated genes (based on limma voom t-value) upon SOX11 knockdown in IMR-32 cells represented in a heatmap. Heatmap color reflects row-wise z-score. B. Top 50 up- and downregulated genes (based on limma voom t-value) upon SOX11 overexpression in SH-EP cells represented in a heatmap. Heatmap color reflects row-wise z-score. C. Gene set enrichment analysis for the differentially expressed genes upon SOX11 knockdown in IMR-32 cells and upon SOX11 overexpression in SH-EP cells shows significant enrichment for genesets involved in cell adhesion, cytoskeleton, axonal outgrowth, epigenetic control, mRNA processing and translation.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Knockdown, Over Expression, Control

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: a. Volcanoplot representing the genes differentially expressed upon SOX11 overexpression in SH-EP (left, blue, adj p value < 0.05, log FC > 1 or < −1) and SOX11 knockdown in IMR-32 (right, blue, adj p value < 0.05, log FC > 0.5 or < −0.5). b . The MsigDB geneset SOX11 target genes (with genes containing one or more binding sites in their promoter) is highly enriched in the downregulated genes upon SOX11 knockdown in IMR-32 cells and in the upregulated genes upon SOX11 overexpression in SH-EP cells, which is validating the RNA-seq data. c. SOX11 and CDKN1A log2 mRNA expression levels upon SOX11 knockdown in IMR-32 cells and SOX11 and MARCKSL1 log2 mRNA expression levels upon SOX11 overexpression in SH-EP cells. Error bars and diamond shape represent respectively the standard deviation and mean of the four biological replicates. Statistical analysis with moderated t-test of Limma Voom (si NTC vs si SOX11 : SOX11 p=6.8e-07, CDKN1A p=6.8e-07; DOX vs UT: SOX11 p=2.8e-15, MARCKSL1 p=5.2e-16). d. Enrichment of the GO geneset "GO nBAF complex" (neuron-specific BAF or SWI/SNF complex) in the upregulated genes upon SOX11 overexpression in SH-EP cells. The expression of the representing leading edge is depicted in heatmaps upon SOX11 overexpression in SH-EP (DOX treated vs Untreated) as well as upon SOX11 knockdown in IMR-32 cells (siSOX11 vs siNTC). e. Enrichment of the GO genesets "GO enhancer binding' and " GO core promoter proximal region DNA binding" in the upregulated genes upon SOX11 overexpression in SH-EP cells. The expression of the representing leading edges depicted in heatmaps. f. Enrichment of the Proneural and Mesenchymal genesets in glioblastoma , the Adrenergic and Mesenchymal genesets in neuroblastoma and the mesenchymal vs NB stage 1 genesets in the down- and up-regulated genes upon SOX11 knockdown in IMR-32 and SOX11 overexpression in SH-EP. g. Enrichment of the GO genesets "GO Cell cell adhesion via plasma membrane adhesion molecules" and "GO Rho guanyl nucleotide exchange factor activity", in the upregulated genes upon SOX11 overexpression in SH-EP cells. The expression of the representing leading edges is depicted in heatmaps.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Over Expression, Knockdown, Binding Assay, RNA Sequencing, Expressing, Standard Deviation, Clinical Proteomics, Membrane, Activity Assay

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: a. Distribution of SOX motif, non-canonical E-box motif ASCL1, and canonical E-box motif MYCN at SOX11 peaks in IMR-32 cells (motifs per bp per peak). Signal is depicted −500 to +500 bp from the peak summit. P-value is determined by Homer (SOX motif p=1e-233, ASCL1 motif p=1e-31, MYCN motif p=1e-28). b. Genome-wide peak and gene annotation distribution (%) (Homer annotation) for the SOX11 peaks called in IMR-32 cells (MACS2, q<0.05). c. Correlation matrix representing the correlation of si SOX11 signature in IMR-32 and SOX11 overexpression signature in SH-EP, SOX11 ChIP-seq targets in IMR-32 (all rank sum activity score) and the ranked expression of SOX11 and affected genes c- MYB , MYCN , SETMAR , MTF2 , SMARCC1 and CDKN1A in a panel of 29 NB cell lines. The size of dots and color represents the correlation coefficient (Spearman correlation), for the correlations with p<0.05. d. Correlation of activity score (rankSum) of the si SOX11 signature, ChIP target signature in IMR-32 and SOX11 mRNA expression levels in the NRC NB tumor cohort ( n = 283, GSE85047) (Left: p=0, r=0.579; middle: p=0, r=0.579; right: p=9.04e-30, r=0.612; Spearman correlation). MYCN status is depicted for every tumor sample. For figure C and D, SOX11 expression was omitted from the si SOX11 signature score to avoid bias. e. Correlation of the activity score (rankSum) of ChIP targets in IMR-32 (top) and the SOX11 regulated mRNAs signature (bottom) in IMR-32 with overall survival in the NRC NB tumor cohort ( n = 283, GSE85047). f. EnrichR analysis ( http://amp.pharm.mssm.edu/Enrichr/ ) for the SOX11 ChIP targets downregulated after SOX11 knockdown and upregulated after SOX11 overexpression. g. SMARCA4, SMARCC1, c-MYB and SOX11 protein levels and loading control Vinculin in an additional clone of SH-EP cells upon SOX11 overexpression over time. h. SOX11, SMARCC1, c-MYB and MYCN protein levels and loading control ACTB in NGP cells upon knockdown of SOX11 using 4 different shRNAs and a non-targeting control (NTC). i. EnrichR analysis ( http://amp.pharm.mssm.edu/Enrichr/ ) for the regions overlapping between the SOX11 and MYCN ChIP targets. For F and I, combined Z-score is depicted based on the multiplication of log p value computed with Fisher exact test and the z-score which is the deviation from the expected rank by the Fisher exact test.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Genome Wide, Over Expression, ChIP-sequencing, Activity Assay, Expressing, Knockdown, Control

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. Overlap (min. overlap = 20 bp) of the SOX11, H3K27ac, H3K4me3 ChIP-seq and ATAC peaks (MACS2 peakcalling qval < 0.05) in the IMR-32 cell line (Fisher test p-value < 2.2e −16 ). B. Binding of SOX11 and MYCN at MTF2 (most significant target), SMARCC1, c-MYB, SMARCE1, SETMAR (2 nd highest significant target), SMARCAD1 and CBX1 in IMR-32 cells and the overlap with H3K27ac, H3K4me3 and ATAC-seq peaks. Signal represents log likelihood ratio for the ChIP signal compared to input signal (RPM normalised). All peaks are called by MACS2 (q<0.05). C. Overlap of SOX11 ChIP-seq targets and genes perturbated upon SOX11 overexpression and SOX11 knockdown and overlap of the SOX11 ChIP-seq targets and the overlapping positively and negatively correlated genes in one or both NB tumor cohorts. Gene annotation for the SOX11 ChIP-seq targets is performed with Homer. D. Coverage of SOX11 ChIP signal for downregulated, upregulated genes and genes not affected (logFC) upon si SOX11 knockdown in IMR-32 cells. Signal is depicted −2000 to +2000 bp distance from the peak summit. E. EnrichR analysis ( http://amp.pharm.mssm.edu/Enrichr/ ) for SOX11 ChIP targets downregulated after SOX11 knockdown and upregulated after SOX11 overexpression. Depicted is the combined Z-score based on the multiplication of log p value computed with Fisher exact test and the z-score which is the deviation from the expected rank by the Fisher exact test (size), as well as the number of genes that overlap with the enriched genesets (color). F. 88 genes signature obtained by the overlap of upregulated genes upon SOX11 knockdown, downregulated genes upon SOX11 overexpression, SOX11 ChIP-seq targets, and positive correlation with SOX11 expression in 2 different NB tumor cohorts (p-value < 0.05). The chromatin modifiers/remodelers CBX1, CBX2, SMARCC1, SMARCAD1, BCL11A and c-MYB are highlighted by an arrow. A color next to each gene represents the involved pathways. G. SMARCA4, SMARCC1, c-MYB and SOX11 and Vinculin protein levels in SH-EP cells upon SOX11 overexpression. H. Heatmap profiles −2 kb and +2 kb around the summit of SOX11 ChIP-seq peaks. The heatmaps represent the ChIP-seq overlap of SOX11 and MYCN peaks in IMR-32, grouped for promoters or enhancers (homer annotation), and ranked according to the sums of the ChIP-seq peak scores across all ChIP-seq peaks in the heatmap.

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: ChIP-sequencing, Binding Assay, Over Expression, Knockdown, Expressing

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: A. ATAC-seq peaks with lost chromatin accessibility upon SOX11 knockdown in CLB-GA cells are annotated by homer as “promoter” or “enhancer”. For both classes overlap with H3K27ac and H3K4me1 is depicted. Motif enrichment of the CRC transcription factors PHOX2B, HAND2, ISL1 and GATA3 is found in the enhancers with lost chromatin accessibility overlapping with H3K27ac and H3K4me1. Heatmap profiles −2 kb and +2 kb around the summit of differential ATAC-seq peaks at enhancers or promoters upon SOX11 knockdown, depicting ATAC peaks with and without SOX11 knockdown (2 biological replicates), and GATA3, HAND2 and PHOX2B ChIP-seq profiles, ranked according to the sums of the peak scores across all peaks in the heatmap. Density profiles are shown representing the average ATAC- or ChIP-seq signal at the presented regions for promotors (orange) and enhancers (blue). B. Average RPKM normalized ATAC-seq reads upon siSOX11 knockdown (siSOX11, orange) as compared to non-targeting control (siNTC, blue) in CLB-GA cells, −0.5Mb and +0.5Mb around the center of adrenergic specific super-enhancers . C. ISL1, GATA3, PHOX2B, TBX2, HAND2 and SOX11 (log2) expression during induced differentiation of hPSC cells along the sympatho-adrenal lineage. Expression levels depicted starting from day 16, upon sorting the cells for SOX10 expression indicating cells committed to truncal neural crest cells, and followed-up during sympatho-adrenal development until day 25

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Knockdown, ChIP-sequencing, Control, Expressing

Journal: bioRxiv

Article Title: SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

doi: 10.1101/2020.08.21.261131

Figure Lengend Snippet: a. Overlap (min. overlap = 20 bp) of enhancers with lost chromatin accessibility upon SOX11 knockdown, and PHOX2B, HAND2 and GATA3 binding in the CLB-GA cell line. Binding has only been taking into account when overlapping for at least 50% with H3K27ac and H3K4me1 in parental CLB-GA cells. b. Heatmap profiles −2 kb and +2 kb around the summit of SOX11 bound enhancers in IMR-32. The heatmaps represent the RPKM signals for ATAC-seq upon SOX11 knockdown (2 biological replicates), GATA3, HAND2, PHOX2B and SOX11 ChIP-seq profiles, ranked according to the sums of the peak scores across all peaks in the heatmap. Density profiles are shown representing the average ATAC- or ChIP-seq signal at the presented region. c. Heatmap profiles 2 kb and +2 kb around the summit of the top 1000 PHOX2B binding sites in CLB-GA. The heatmaps represent the RPKM signals for ATAC-seq upon SOX11 knockdown (2 biological replicates), GATA3, HAND2, PHOX2B and SOX11 ChIP-seq profiles, ranked according to the sums of the peak scores across all peaks in the heatmap. Density profiles are shown representing the average ATAC- or ChIP-seq signal at the presented motifs. d. ATAC-seq profiles upon SOX11 knockdown and control cells, as well as ChIP-seq profiles for SOX11 and MYCN at super-enhancer regions in adrenergic cells (RPKM normalised). e. ISL1, GATA3, PHOX2B, TBX2, HAND2 and SOX11 (log2) expression during induced differentiation of hPSC cells along the sympatho-adrenal lineage. Expression levels depicted starting from day 16, when cells are sorted by SOX10 expression levels indicating cells committed to truncal neural crest cells, and followed-up during SAP development stadia until day 25, for three additional replicates (differentiation tracks)

Article Snippet: For SOX11 inducible overexpression, the OriGene vector SC303275 containing the cDNA of SOX11 was amplified by PCR and the obtained fragment was gel purified and ligated into the opened NdeI site of response vector pLVX-TRE2G-Zsgreen1 (Takara, cat#631353) producing pLVX-TRE3G-Zsgreen1-IRES-hSOX11.

Techniques: Knockdown, Binding Assay, ChIP-sequencing, Control, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 1. Sox11 plasmid transfection significantly improved the the lung wet/dry weight ratio, cell numbers and protein and RAGE levels in BALF induced by, and FAK antagonist prevented the improvement of Sox11 plasmid transfection. The detail illustration please refer to the Supplementary Material for this and following Figs).

Article Snippet: Mouse Sox11 cDNAs (Origene) were sequenced and subcloned into the pCMV-IRES-EGFP plasmid (Addgene, Cambridge, MA, US), and the IRES-EGFP sequence was removed to create pCMV-Sox11 Please cite this article as: M. Fang et al., Transfection of Sox11 plasmid alle and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc following the manufacturer's instructions.

Techniques: Plasmid Preparation, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 2. The lung injury and apoptosis were after The transfection of Sox11 plasmid significantly improved pulmonary pathology and apoptosis induced by HMV and the FAK antagonist prevented the improvement of Sox11 plasmid transfection on the lung injury.

Article Snippet: Mouse Sox11 cDNAs (Origene) were sequenced and subcloned into the pCMV-IRES-EGFP plasmid (Addgene, Cambridge, MA, US), and the IRES-EGFP sequence was removed to create pCMV-Sox11 Please cite this article as: M. Fang et al., Transfection of Sox11 plasmid alle and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc following the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 3. HMV significantly downregulated the expression of Sox11 and FAK in mRNA and protein levels, and Sox11 plasmid transfection significantly restored the downregulation in the HMV mice. FAK antagonist blocked the restore of FAK in Sox11 plasmid transfection mice.

Article Snippet: Mouse Sox11 cDNAs (Origene) were sequenced and subcloned into the pCMV-IRES-EGFP plasmid (Addgene, Cambridge, MA, US), and the IRES-EGFP sequence was removed to create pCMV-Sox11 Please cite this article as: M. Fang et al., Transfection of Sox11 plasmid alle and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc following the manufacturer's instructions.

Techniques: Expressing, Plasmid Preparation, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 4. The immunoreactivity of Sox11 and FAK was significantly decreased after HMV, and the decreases were effectively restored by the transfection of Sox11 plasmid. The FAK antagonist blocked the restore of FAK immunoreactivity in Sox11 plasmid transfection mice.

Article Snippet: Mouse Sox11 cDNAs (Origene) were sequenced and subcloned into the pCMV-IRES-EGFP plasmid (Addgene, Cambridge, MA, US), and the IRES-EGFP sequence was removed to create pCMV-Sox11 Please cite this article as: M. Fang et al., Transfection of Sox11 plasmid alle and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc following the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation

Journal: Genome biology

Article Title: Transcriptional analysis of metastatic hormone-naïve prostate cancer primary tumor biopsies reveals a relevant role for SOX11 in prostate cancer cell dissemination.

doi: 10.1186/s13059-025-03623-5

Figure Lengend Snippet: Fig. 4 Proof-of-concept study of SOX11 as a metastatic driver in prostate cancer. A Waterfall plot representing the differential expression of transcription factors in mHNPC vs. LPC bulk RNA-Seq in discovery and validation cohorts. The position of SOX11 is highlighted. B Representative western blot in DU145 cells showing ectopic 3HA-SOX11 expression (n = 3). C Evaluation of mRNA abundance in the indicated genes by quantitative real-time PCR upon ectopic 3HA-SOX11 expression (n = 3–6). One sample t-test. D. Experimental design of the orthotopic injection of DU145 cells transduced with control and 3HA-SOX11-overexpressing lentiviral vectors into nude mice (n = 5 for 3HA, n = 6 for 3HA-SOX11). E Left, dot plot of lumbar lymph node (LLN) metastatic burden (day 51). One-tailed, Mann Whitney test. Middle, stacked bar plot of LLN metastasis appearance. One-tailed, Fisher test. Right, representative bioluminescence images of LLN metastasis. F REACTOME pathway enrichment analyses of the genes upregulated upon SOX11 overexpression. G Violin plots showing the levels of a SOX11-activity signature in the discovery and validation bulk RNA-Seq datasets. P-values from Wilcoxon test (two-tailed test for discovery and one-tailed for validation datasets, respectively). H Violin plots showing the levels of a signature composed of genes encoding for purple module ligands identified in single cell RNA-Seq and correlated with SOX11 in the discovery and validation bulk RNA-Seq datasets. P-values from Wilcoxon test (two-tailed test for discovery and one-tailed for validation datasets, respectively). I Hazard ratio (HR) and p-value of a signature based on the ligands used in H when analyzing as endpoint biochemical recurrence in the indicated datasets and comparing patients within quartiles 1 and 4. A representative Kaplan–Meyer curve is shown

Article Snippet: Primary antibodies for HA (Cell Signalling Technologies, 3724), SOX11 (Cell Signalling Technologies, 58,207), and HSP90 (Cell Signalling Technologies, 4874S) were used at a 1:1000 dilution.

Techniques: Quantitative Proteomics, RNA Sequencing, Biomarker Discovery, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Injection, Transduction, Control, One-tailed Test, MANN-WHITNEY, Over Expression, Activity Assay, Two Tailed Test

Journal: Experimental Animals

Article Title: Dual specificity protein phosphatase 5, transcriptionally inhibited by SRY-box transcription factor 11, inhibits T helper 2 differentiation in CD4 + T cells: a promising therapeutic target for allergic rhinitis

doi: 10.1538/expanim.25-0076

Figure Lengend Snippet: SOX11 inhibited the promoter activity and thereby decreasing the transcription of Dusp5 . (A) Representative images of immunofluorescence double staining of SOX11 (red) and CD4 (green) in nasal mucosa tissues (400×). (B and C) The potential binding site of SOX11 in the promoter region of Dusp5 and the binding motif of SOX1 were predicted by JASPAR database. (D) Activities of the Dusp5 promoter of different lengths were detected by dual-luciferase reporter assay. The data were shown as the mean ± SD. * P -value <0.05; *** P -value <0.001. SOX11, SRY-box transcription factor 11; DUSP, dual specificity protein phosphatase.

Article Snippet: Then, the sections were co-incubated with antibody against DUSP5 (1: 200; Abcam, Cambridge, UK), SOX11 (1: 100; ABclonal, Wuhan, China), or CD4 (1: 50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C.

Techniques: Activity Assay, Immunofluorescence, Double Staining, Binding Assay, Luciferase, Reporter Assay

Journal: Experimental Animals

Article Title: Dual specificity protein phosphatase 5, transcriptionally inhibited by SRY-box transcription factor 11, inhibits T helper 2 differentiation in CD4 + T cells: a promising therapeutic target for allergic rhinitis

doi: 10.1538/expanim.25-0076

Figure Lengend Snippet: DUSP5 overexpression reversed SOX11 overexpression-induced Th2 differentiation. (A and B) The expression of SOX11 and DUSP5 in Th2 cells. (C) Levels of IL-5 and IL-13 in the cell culture supernatant. (D) After being stimulated with PMA and ionomycin, the expression of IL-4 in CD4+ T cells was analyzed using flow cytometry. The data were shown as the mean ± SD. * P -value <0.05; ** P -value <0.01; *** P -value <0.001. SOX11, SRY-box transcription factor 11; DUSP, dual specificity protein phosphatase; PMA, phorbol myristate acetate.

Article Snippet: Then, the sections were co-incubated with antibody against DUSP5 (1: 200; Abcam, Cambridge, UK), SOX11 (1: 100; ABclonal, Wuhan, China), or CD4 (1: 50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C.

Techniques: Over Expression, Expressing, Cell Culture, Flow Cytometry

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 1. Sox11 plasmid transfection significantly improved the the lung wet/dry weight ratio, cell numbers and protein and RAGE levels in BALF induced by, and FAK antagonist prevented the improvement of Sox11 plasmid transfection. The detail illustration please refer to the Supplementary Material for this and following Figs).

Article Snippet: Mice in HMV group were received HMV for 4 h. Mice in Sox11 plasmid and Sox11 plasmid þ FAK antagonist groups were first administrated with Sox11 plasmid only (10 mg/kg, Origene, USA) or co-administrated with Sox11 plasmid and the antagonist of FAK (PF562271, 25 mg/kg, Houston, USA), respectively, and then received HMV for 4 h. Meanwhile, a vehicle group for Sox11 plasmid and FAK antagonist was designed, in which vector of Sox11 plasmid or DMSO, the solvent of FAK antagonist [8], was administrated instead of the plasmid or antagonist, respectively.

Techniques: Plasmid Preparation, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 2. The lung injury and apoptosis were after The transfection of Sox11 plasmid significantly improved pulmonary pathology and apoptosis induced by HMV and the FAK antagonist prevented the improvement of Sox11 plasmid transfection on the lung injury.

Article Snippet: Mice in HMV group were received HMV for 4 h. Mice in Sox11 plasmid and Sox11 plasmid þ FAK antagonist groups were first administrated with Sox11 plasmid only (10 mg/kg, Origene, USA) or co-administrated with Sox11 plasmid and the antagonist of FAK (PF562271, 25 mg/kg, Houston, USA), respectively, and then received HMV for 4 h. Meanwhile, a vehicle group for Sox11 plasmid and FAK antagonist was designed, in which vector of Sox11 plasmid or DMSO, the solvent of FAK antagonist [8], was administrated instead of the plasmid or antagonist, respectively.

Techniques: Transfection, Plasmid Preparation

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 3. HMV significantly downregulated the expression of Sox11 and FAK in mRNA and protein levels, and Sox11 plasmid transfection significantly restored the downregulation in the HMV mice. FAK antagonist blocked the restore of FAK in Sox11 plasmid transfection mice.

Article Snippet: Mice in HMV group were received HMV for 4 h. Mice in Sox11 plasmid and Sox11 plasmid þ FAK antagonist groups were first administrated with Sox11 plasmid only (10 mg/kg, Origene, USA) or co-administrated with Sox11 plasmid and the antagonist of FAK (PF562271, 25 mg/kg, Houston, USA), respectively, and then received HMV for 4 h. Meanwhile, a vehicle group for Sox11 plasmid and FAK antagonist was designed, in which vector of Sox11 plasmid or DMSO, the solvent of FAK antagonist [8], was administrated instead of the plasmid or antagonist, respectively.

Techniques: Expressing, Plasmid Preparation, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Transfection of Sox11 plasmid alleviates ventilator-induced lung injury via Sox11 and FAK.

doi: 10.1016/j.bbrc.2019.03.041

Figure Lengend Snippet: Fig. 4. The immunoreactivity of Sox11 and FAK was significantly decreased after HMV, and the decreases were effectively restored by the transfection of Sox11 plasmid. The FAK antagonist blocked the restore of FAK immunoreactivity in Sox11 plasmid transfection mice.

Article Snippet: Mice in HMV group were received HMV for 4 h. Mice in Sox11 plasmid and Sox11 plasmid þ FAK antagonist groups were first administrated with Sox11 plasmid only (10 mg/kg, Origene, USA) or co-administrated with Sox11 plasmid and the antagonist of FAK (PF562271, 25 mg/kg, Houston, USA), respectively, and then received HMV for 4 h. Meanwhile, a vehicle group for Sox11 plasmid and FAK antagonist was designed, in which vector of Sox11 plasmid or DMSO, the solvent of FAK antagonist [8], was administrated instead of the plasmid or antagonist, respectively.

Techniques: Transfection, Plasmid Preparation

Journal: Animals : an Open Access Journal from MDPI

Article Title: A Functional Single-Nucleotide Polymorphism Upstream of the Collagen Type III Gene Is Associated with Catastrophic Fracture Risk in Thoroughbred Horses

doi: 10.3390/ani14010116

Figure Lengend Snippet: SOX11 expression. SOX11 is expressed in Thoroughbred skin fibroblasts pre and post differentiation into osteoblast-like cells. There are no significant differences in expression between cells derived from high- and low-risk horses. Expression is shown relative to the housekeeping gene. Error bars represent the s.e.m of cells derived from three individual horses. Cells were at passages 2–6.

Article Snippet: First, 1 × 10 6 skin fibroblasts (from one Thoroughbred and two Welsh Mountain ponies as this experiment is independent of breed) were suspended in 400 μL of standard culture media lacking penicillin/streptomycin and either 20 μg of pCMV6-AC-GFP human SOX11 (NM_003108; Origene, Rockville, MD, USA) or pCAG-eGFP [ ], which was a gift from Peter Andrews (University of Sheffield, Sheffield, UK).

Techniques: Expressing, Derivative Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: A Functional Single-Nucleotide Polymorphism Upstream of the Collagen Type III Gene Is Associated with Catastrophic Fracture Risk in Thoroughbred Horses

doi: 10.3390/ani14010116

Figure Lengend Snippet: ( A ) SOX11 stable modulation results in significant changes in COL3A1 expression: ( Ai ) Stable transfection of a SOX11 overexpression plasmid (pSOX11) results in a significant increase in SOX11 expression in equine skin fibroblasts (one Thoroughbred, two ponies), pre and post osteogenic differentiation, compared with control cells (pGFP). ( Aii ) This results in a significant increase in COL3A1 expression in the undifferentiated cells (pre osteogenic culture). ( B ) Lentiviral expression of a shRNA to SOX11 reduces SOX11 ( Bi ) and COL3A1 ( Bii ) expression in undifferentiated skin fibroblasts (three Thoroughbred, one pony). Expression is shown relative to the housekeeping gene. Error bars represent the s.e.m from cells derived from three or four different donors. * p < 0.05. Cells between passages 5 and 10 were used in these experiments.

Article Snippet: First, 1 × 10 6 skin fibroblasts (from one Thoroughbred and two Welsh Mountain ponies as this experiment is independent of breed) were suspended in 400 μL of standard culture media lacking penicillin/streptomycin and either 20 μg of pCMV6-AC-GFP human SOX11 (NM_003108; Origene, Rockville, MD, USA) or pCAG-eGFP [ ], which was a gift from Peter Andrews (University of Sheffield, Sheffield, UK).

Techniques: Expressing, Stable Transfection, Over Expression, Plasmid Preparation, Control, shRNA, Derivative Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: A Functional Single-Nucleotide Polymorphism Upstream of the Collagen Type III Gene Is Associated with Catastrophic Fracture Risk in Thoroughbred Horses

doi: 10.3390/ani14010116

Figure Lengend Snippet: The region upstream of COL3A1 containing the SNP can bind to a nuclear protein. EMSA revealed that a probe containing either the wild-type (TT) or variant (GG) allele was capable of binding to a nuclear protein as seen by the shift (arrow). The use of an unlabelled competitor probe prevented this shift from occurring. The addition of an antibody to SOX11 did not result in a detectable supershift. Image representative of results observed using nuclear extracts from fibroblasts isolated from two different ponies. The boxed area represents a technical control for the assay, carried out using a biotin-labelled probe for Epstein–Barr Nuclear Antigen (EBNA, labelled E in the figure), an unlabelled probe, and an EBNA protein extract. The image is of the entire full-size blot and is not cropped.

Article Snippet: First, 1 × 10 6 skin fibroblasts (from one Thoroughbred and two Welsh Mountain ponies as this experiment is independent of breed) were suspended in 400 μL of standard culture media lacking penicillin/streptomycin and either 20 μg of pCMV6-AC-GFP human SOX11 (NM_003108; Origene, Rockville, MD, USA) or pCAG-eGFP [ ], which was a gift from Peter Andrews (University of Sheffield, Sheffield, UK).

Techniques: Variant Assay, Binding Assay, Isolation, Control