Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) Heatmaps depict EWS-FLI1, H3K4me1 and H3K27ac signal intensities for 1604 EWS-FLI1-bound distal regulatory elements. Rows: 10 kb regions, centered on EWS-FLI1 peaks, ranked by overall signal intensities of H3K4me1 and H3K27ac. (B) Heatmaps depict EWS-FLI1 and H3K4me3 signals for 181 EWS-FLI1-peaks overlapping with transcriptional start sites (TSS). Rows: 10 kb regions, centered on EWS-FL1 peaks, ranked by overall signal intensities of H3K4me3. EWS-FLI1-binds to enhancers with variable levels of activity as demonstrated by the presence of the H3K4me1 mark and different levels of the H3K27ac activation mark. In contrast EWS-FLI1 is primarily found at active promoters. (C) Composite plots showing average levels of H3K27me3 signals at EWS-FLI1 binding sites (left), compared to genome-wide signals at H3K27me3 peaks (right). (D) Examples of active distal regulatory elements near known EWS-FLI1 target genes in Ewing sarcoma cell lines (A673 and SKMNC) and a primary tumor. Tracks show EWS-FLI1, H3K27ac and H3K4me1 signals. EWS-FLI1 binding is highlighted in gray. See also Figure S1 and Table S1.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Activity Assay, Activation Assay, Binding Assay, Genome Wide

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) Heatmaps (left) and composite plots (middle) depicting H3K27ac and p300 signal intensity changes across EWS-FLI1 peaks after EWS-FLI1 knock-down in SKNMC cells at indicated time points. Binding sites are classified as repressed if EWS-FLI1 depletion results in increased H3K27ac and p300 signals (top, 330 sites, 1.5 fold increase in H3K27ac), or activated if depletion results in decreases in H3K27ac and p300 (bottom, 1011 sites, 1.5 fold decrease in H3K27ac). Right: de novo motif analysis of repressed peaks shows strong enrichment for the canonical ETS factor family motifs (p = 1e−129, top); activated peaks show enrichment for consecutive GGAA repeat elements (p = 1e−878, bottom). (B) Signal tracks for representative repressed binding sites (ENC1 and RAB3GAP2) in SKNMC cells. EWS-FLI1, H3K27ac and p300 signals for shGFP or shFLI1 infected cells are shown. The genomic sequence for the EWS-FLI1 binding site near ENC1 is provided (single GGAA). (C) Signal tracks for representative activated binding sites (NKX2-2 and NPY1R) as in (B). The genomic sequence for the EWS-FLI1 binding site near NKX2-2 is provided (GGAA repeats). Areas of EWS-FLI1 binding are highlighted in gray. These data suggest that repression and activation of EWS-FLI1 bound sites rely on two distinct chromatin remodeling mechanisms, dictated by the underlying genomic sequence and the differential recruitment of p300. See also Figure S2.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Binding Assay, Infection, Sequencing, Activation Assay

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) Boxplots for DNAseI signals at activated (top) or repressed (bottom) EWS-FLI1 binding sites across 112 cell lines profiled by ENCODE. SKNMC cells are shown in red. (B) Conservation scores (PhastCons) in 100 vertebrate species for 2 kb intervals centered on activated or repressed EWS-FLI1 binding sites. (C) Left: Comparison of H3K27ac changes at EWS-FLI1 binding sites after introduction of EWS-FLI1 in MSCs or after EWS-FLI1 depletion in SKNMC cells. Activated EWS-FLI1 binding sites are boxed with a dashed line. Right: Boxplots of H3K27ac (top) and H3K4me1 (bottom) signal intensities at 1011 EWS-FLI1 activated sites after introduction of EWS-FLI1 in MSCs (blue) compared to an empty vector control (black). Signals for both enhancer marks are significantly induced following EWS-FLI1 expression. (D) Composite plots of WDR5 and H3K4me1 signals at activated EWS-FLI1 binding sites in MSCs expressing EWS-FLI1 or infected with an empty vector. Signals in SKMNC cells are shown on the right panel for comparison. (E) Heatmaps depict signals for ATAC-seq, WDR5, H3K4me1 and H3K27ac at activated binding sites as in C, either from empty vector infected or EWS-FLI1-expressing MSCs. (rows: ATAC-seq 2 kb region, WDR5-H3K4me1-H3K27ac 10 kb regions, centered on EWS-FLI1 peaks). (F) Signal tracks for FLI1, H3K4me1, H3K27ac, WDR5 and ATAC-seq at the NKX2-2 locus in SKNMC cells, and MSCs expressing EWS-FLI1 (E-F) or empty vector control (Co). EWS-FLI1 expression in MSCs leads to nucleosomal rearrangement, WDR5 recruitment, and de novo deposition of both enhancer marks H3K4me1 and H3K27ac, recapitulating the open active chromatin architecture of SKNMC cells. (G-H) 3C-qPCR analysis of long-distance interactions between the NKX2-2 promoter and the corresponding EWS-FLI1-bound distal regulatory element in SKNMC (G) and primary mesenchymal stem cells (H). A strong interaction is present between the distal regulatory element and the promoter of NKX2-2 in SKNMC cells. No significant interaction is observed in MSCs until introduction of EWS-FLI1 leads to DNA looping (H) to produce a conformation similar to SKNMC cells. The human NKX2-2 locus is depicted above each graph. The x-axes represent distances (kb) from the NKX2-2 promoter. A red arrow denotes the HindIII fragment serving as anchor, black and blue arrows denote the analyzed HindIII fragments. P: promoter; E: enhancer. Error bars represent standard deviations. See also Figure S3.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Binding Assay, Plasmid Preparation, Expressing, Infection

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) Boxplots of H3K27ac signal intensities at distal elements corresponding to EWS-FLI1 peaks (repressed sites = blue, activated sites = black) in H1 embryonic stem cells, H1-derived NPCs (neural progenitor cells), H1-derived MSCs, bone marrow-derived MSCs, osteoblasts, skeletal muscle myoblasts (HSMM) and dermal fibroblasts (NHDF). Repressed EWS-FLI1 bound distal elements in Ewing sarcoma are active in normal mesenchymal cell types but not in H1 ES cells or H1-derived NPCs. (B) Composite plots show EWS-FLI1 (left) and ELF1 (right) signal intensities for 330 repressed EWS-FLI1 binding sites upon EWS-FLI1 depletion in SKNMC cells. (C) Heatmaps depict signals for EWS-FLI1, ELF1 and p300 at the same repressed sites (rows: 2 kb regions centered on EWS-FL1 peaks). ELF1 binding is observed at many of these sites upon EWS-FLI1 depletion. (D) Signal tracks for EWS-FLI1, H3K27ac, p300 and ELF1 at the ENC1 locus in SKMNC cells. After EWS-FLI1 depletion ELF1 binding leads to p300 recruitment and enhancer activation. Areas of EWS-FLI1 binding are highlighted in gray. See also Figure S4s.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Derivative Assay, Binding Assay, Activation Assay

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) Box-plots show Z-scores for gene expression changes vs Z-scores for H3K27ac chromatin changes after EWS-FLI1 knock-down in SKNMC and A673 cells (48 hr). Z-scores provide a measure of effect size and consistency between cell lines. The nearest expressed genes in SKNMC and A673 cells were assigned to each binding site. (B) Heatmaps depict fold changes in H3K27ac and gene expression in A673 and SKNMC Ewing cells after EWS-FLI1 knock-down (48 hr). Genes were ranked by the combined significance of H3K27ac and gene expression changes (average z-score). The top 100 activated or repressed enhancer binding sites and genes are shown in the heatmap and the top 10 annotated genes are listed on the right. (C) Track signals for FLI1, H3K27ac and RNA-seq in SKNMC after EWS-FLI1 depletion (96 hr) identify an active regulatory element distal to VRK1 (top). The same enhancer element is present in primary Ewing tumors (middle), and is generated de novo by EWS-FLI1 expression in MSCs (bottom). P: promoter; E: enhancer. (D-E) 3C-qPCR analysis of long-distance interactions between the VRK1 promoter and the corresponding EWS-FLI1-bound distal regulatory element in SKNMC (D) and A673 cells (E). The human VRK1 locus is depicted below each graph. The x-axes represent distances (kb) from the VRK1 promoter. Red arrow denotes the HindIII fragment serving as anchor, black and blue arrows denote the analyzed HindIII fragments. P: promoter; E: enhancer. Error bars represent standard deviations. See also Figure S5, Table S3 and S4.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Expressing, Binding Assay, RNA Sequencing Assay, Generated

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: (A) VRK1 is expressed in the majority of Ewing sarcoma cells, as assessed by immunohistochemistry of primary tumors (magnification: 400x, scale bar: 50 uM). (B) Left: VRK1 mRNA expression in A673 and SKNMC cells after EWS-FLI1 depletion (shFLI1) compared to control (shGFP). Right: VRK1 mRNA expression in MSCs after introduction of EWS-FLI1 (pLIV EWS-FLI1) compared to control cells (pLIV empty). Error bars represent standard deviations. (C) Proliferation rates and relative apoptosis (D) of a panel of tumor cell lines after VRK1 knock-down compared to control (shGFP). Error bars represent the standard deviation of three replicates. Ewing sarcoma cells display selective high sensitivity toward VRK1 depletion, compared to Saos-2 (osteosarcoma) and HeLa cells. (E-F) VRK1 depletion markedly reduces tumor growth in immunocompromised mice, as assessed by tumor weight (E) and volume (F) 3 weeks after subcutaneous injection of control or VRK1-depleted SKNMC cells. Error bars represent standard deviations.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Immunohistochemistry, Expressing, Standard Deviation, Injection

Journal: Cancer cell

Article Title: EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

doi: 10.1016/j.ccell.2014.10.004

Figure Lengend Snippet: Schematic illustrating the two distinct chromatin remodeling mechanisms underlying EWS-FLI1 divergent transcriptional activity: enhancer induction and activation (upper panel) with recruitment of WDR5 and p300 at GGAA repeats, and enhancer repression (lower panel) with displacement of endogenous ETS transcription factors and p300 at single GGAA canonical ETS motifs.

Article Snippet: Primary antibodies used for Western blotting were polyclonal rabbit anti-FLI1 (Santa Cruz, sc-356, 1:500 dilution), polyclonal rabbit anti-VRK1 (Santa Cruz, 1F6, 1:500 dilution) and monoclonal mouse anti-PARP (Santa Cruz, sc-8007, 1:500 dilution).

Techniques: Activity Assay, Activation Assay

Journal: Journal of Virology

Article Title: Endothelial Cell Infection by Guinea Pig Cytomegalovirus Is a Lytic or Persistent Infection Depending on Tissue Origin but Requires Viral Pentamer Complex and pp65 Tegument Protein

doi: 10.1128/jvi.00831-22

Figure Lengend Snippet: Characterization of guinea pig aorta-derived endothelial (EndoC) cells by immunostaining and Western blot analysis. The EndoC cell line was verified as endothelial by evaluation for specific endothelial cell markers by IHC staining (i and ii) or by Western blotting (iii to viii) compared to GPL fibroblasts. (i) IHC staining for the endothelial cell marker vWF on EndoC cell lines (A and B) or GPL cells (C and D) carried out in six-well plates. Individual bright-field images of cells stained with rabbit anti-vWF (Abcam) plus anti-rabbit IgG-HRP (Vectastain) (A and C) or secondary-antibody-only negative control (B and D). Magnification, ×10. (ii) IHC staining for the endothelial cell marker FLI1 of EndoC cell lines (A and B), or GPL fibroblasts (C and D) carried out in six-well plates. Individual bright-field images of cells stained with rabbit anti-FLI1 (Antibodies-online) plus anti-rabbit IgG-HRP (Vectastain) (A and C) or secondary-antibody-only negative control (B and D). Magnification, ×10. (iii to viii) Western blot analysis of total cell lysate from various guinea pig cell lines for (iii) PECAM1 (CD31), (iv) FLI1, (vi) CACNA1S, and (vii) SMA. (v and viii) Lane loading control β-actin. Lanes contain cell lysates: (1) EndoC clone 1 (EndoC-c1), (2) EndoC clone 2 (EndoC-c2), (3) REPI cells, and (4) GPL fibroblasts. Western blots were carried out as described in Materials and Methods.

Article Snippet: Individual bright-field images of cells stained with rabbit anti-FLI1 (Antibodies-online) plus anti-rabbit IgG-HRP (Vectastain) (A and C) or secondary-antibody-only negative control (B and D).

Techniques: Derivative Assay, Immunostaining, Western Blot, Immunohistochemistry, Marker, Staining, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: Establishment of stable DLD-1 cell lines with inducible EWSR1/FLI1 , EWSR1/FLI1-T79A , and EWSR1/FLI1-T79D . A , Schematic of EWSR1/FLI1, EWSR1/FLI1-T79A , and EWSR1/FLI1-T79D constructs. Tet-on TA: Tet-on Transactivation, Tet Pro: Tet-on promoter, Pur: Puromycin resistance gene. B , The expression of EWSR1/FLI1-mCherry , EWSR1/FLI1-T79A-mCherry , and EWSR1/FLI1-T79D-mCherry in Dox-treated (Dox+) cells and untreated (Dox-) cells was verified by western blot using anti-FLI1 and anti-β-tubulin antibodies. E/F: EWSR1/FLI1. C , Representative images of EWSR1/FLI1-mCherry, EWSR1/FLI1-T79A-mCherry, and EWSR1/FLI1-T79D-mCherry expression ( red ) with DAPI ( blue ) obtained from untreated (Dox-) and treated (Dox+) cells. Scale bar = 10um.

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Construct, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: Localization of EWSR1/FLI1 on chromosomes during early mitosis requires Thr 79 . DLD-1 cells with inducible EWSR1/FLI1 and EWSR1/FLI1-T79A were induced with Dox and synchronized in mitosis using thymidine ( A and B , Fig. S1 A ) or thymidine/nocodazole ( C and D , Fig. S1 B ). A , Cells were subjected to immunocytochemistry using anti-mCherry primary and Alexa Fluor 594 conjugated secondary antibodies ( red ), and DNA was stained with DAPI ( blue ). Single Z-sections of cells were captured and representative merged images of mCherry and DAPI signals (top; red and blue ) and mCherry signal alone (bottom; black and white ) show the localization of EWSR1/FLI1 foci on the chromosomes, compared with EWSR1/FLI1-T79A localization in the cytoplasm. The backgrounds of the images were adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. The edge of rotated image is indicated by thin line. Scale bar = 10um. B , Single Z-section images of EWSR1/FLI1 or EWSR1/FLI1-T79A expressing cells were photodocumented, and the percentages of the cells that displayed the localization of either EWSR1/FLI1 or EWSR1/FLI1-T79A on the chromosome was scored. E/F: EWSR1/FLI1, T79A: EWSR1/FLI1-T79A, Pro: Prophase, Meta: Metaphase, Ana: Anaphase, Telo: Telophase, and Cyto: Cytokinesis. C , Lysates from whole cells and cytoplasm and chromosome fractions were extracted and subjected to western blot using anti-FLI1 (top panel), anti-β-tubulin (middle panel), and anti-H2A antibodies (bottom). The image was cropped, the color was inverted using the “Invert” function of Photoshop software, and the background was adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. D , Relative intensity of the EWSR1/FLI1 and EWSR1/FLI1-T79A (normalized to Histone H2A) bands from the chromosome sample. (n = 3 experiments). ∗∗ p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Immunocytochemistry, Staining, Expressing, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79 is required for colocalization of EWSR1/FLI1 with Aurora B during metaphase . The EWSR1/FLI1 and EWSR1/FLI1-T79A cell lines were synchronized in mitosis using thymidine ( Fig. S1 A ) and treated with (Dox+), followed by immunocytochemistry using anti-mCherry primary and Alexa Fluor 594 conjugated secondary ( red ), and anti-Aurora B primary and Alexa Fluor 488 conjugated secondary ( green ) antibodies. A , Representative images of single Z-sections of cells stained with mCherry/Aurora B/DAPI (left), mCherry (middle), Aurora B (middle), and merged images from mCherry/Aurora B (White represents the areas with colocalized Aurora B ( green ) and mCherry ( red ) signals) (right panel). Scale bar = 10um. B , The Pearson Coefficient of Covariance for Aurora B ( green ) and mCherry ( red ) signals in Dox-treated metaphase cells is significantly higher for cells expressing EWSR1/FLI1 (n = 11 cells) than for cells expressing EWSR1/FLI1-T79A (n = 12 cells). ∗∗ p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Immunocytochemistry, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79 is required for mislocalization of Aurora B at the midzone . A , Representative images of anaphase cells. ]: area with sparse Aurora B signal. Scale bar = 10um. B , The percentage of cells that display aberrant Aurora B localization was increased in cells that express EWSR1/FLI1 but not in cells that express EWSR1/FLI1-T79A (50 anaphase cells per sample, n = 3 experiments). ∗ p < 0.05 and ∗∗ p < 0.01 (ANOVA one way Tukey HSD).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79 is required for the induction of aneuploidy . The stable lines were induced with Dox to express either EWSR1/FLI1 or EWSR1/FLI1-T79A (controls left untreated) and subjected to a metaphase chromosome spread protocol. A , Schematic for the induction of EWSR1/FLI1 and EWSR1/FLI1-T79A using doxycycline and metaphase synchronization using colcemid. B , Representative images of chromosomes from induced cells expressing EWSR1/FLI1 or EWSR1/FLI1-T79A and uninduced cells. (EWSR1/FLI1 Dox-; 46 chromosomes, EWSR1/FLI1 Dox+; 44 chromosomes, EWSR1/FLI1-T79A Dox-; 46 chromosomes, EWSR1/FLI1-T79A Dox+; 46 chromosomes). The color of the chromosome images was inverted using the “Invert” function of Photoshop software, and the background intensity was adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. Scale bar = 10um. C , The percentage of cells that displayed aberrant numbers of chromosomes was increased in cells expressing EWSR1/FLI1 . (20 cells per sample, n = 3 experiments). ∗∗ p < 0.01, NS: not significant (ANOVA one way with post-hoc Tukey HSD). D , The percentage of cells per sample group containing each chromosome number over the full range of chromosome numbers observed in all cells. Cells expressing EWSR1/FLI1 have the greatest percentage of nondiploid chromosome numbers.

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79D localizes on the chromosomes during early mitosis . The stable lines were induced with Dox to express either EWSR1/FLI1 or EWSR1/FLI1-T79D , and the cells were mitotically synchronized by thymidine ( A and B , Fig. S1 A ) or by thymidine/nocodazole ( C and D , Fig. S1 B ). A , Immunocytochemistry was performed using anti-mCherry primary and Alexa Fluor 596 conjugated secondary antibodies ( red ), and DNA was stained with DAPI ( blue ). The backgrounds of single Z-section images of cells were adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. The merged images ( top ) and mCherry signal ( bottom ) display the localization of EWSR1/FLI1 and EWSR1/FLI1-T79D on the chromosomes. Scale bar = 10um. B , The percentages of cells that displayed localization of either EWSR1/FLI1 or EWSR1/FLI1-T79D on the chromosomes at different phases of mitosis. E/F: EWSR1/FLI1, T79D: EWSR1/FLI1-T79D, Pro: Prophase, Meta: Metaphase, Ana: Anaphase, Telo: Telophase, Cyto: Cytokinesis. n: Total number of cells that were scored. C , The lysates were extracted using the same protocol shown in Figure 2 C , and it were subjected to western blot using anti-FLI1 ( top panel ), anti-β-tubulin ( middle panel ), and anti-H2A antibodies ( bottom panel ). The images were cropped, and color was inverted using the “Invert” function of Photoshop software, and the background was adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. D , Relative intensity of the EWSR1/FLI1 and EWSR1/FLI1-T79D bands (normalized to Histone H2A) from the western blot of the chromosome sample. (n = 3 experiments). ∗∗ p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Immunocytochemistry, Staining, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79D display colocalization with Aurora B during metaphase . EWSR1/FLI1 and EWSR1/FLI1-T79D expressing cells (Dox+) and control (Dox-) cells were synchronized in mitosis using thymidine ( Fig. S1 A ), and subjected to immunocytochemistry using anti-mCherry primary and Alexa Fluor 594 ( red ), and anti-Aurora B primary and Alexa Fluor 488 conjugated secondary antibodies ( green ). A , Merged single Z-section images from mCherry/Aurora B/DAPI ( left ), with mCherry ( middle ), with Aurora B ( middle ), and merged images from mCherry/Aurora B ( white represents the areas with colocalized Aurora B ( green ) and mCherry ( red ) signals) ( right panel ). Scale bar = 10um. B , The Pearson Coefficient of covariance for Aurora B ( green ) and mCherry ( red ) signals in Dox-treated metaphase cells. There is significant difference between cells expressing EWSR1/FLI1 and cells expressing EWSR1/FLI1-T79D. ∗∗ p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Expressing, Control, Immunocytochemistry

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79D promotes mislocalization of Aurora B at the midzone . A , Representative images of anaphase cells. [: area with unevenly distributed Aurora B signal. Scale bar = 10um. B , The percentage of cells that display aberrant Aurora B localization was higher in cells that express EWSR1/FLI1-T79D (50 anaphase cells per sample, n = 3 experiments) than in control cells. ∗ p < 0.05 and ∗∗ p < 0.01 (ANOVA one way with post-hoc Tukey HSD).

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: EWSR1/FLI1-T79D promotes the induction of aneuploidy . EWSR1/FLI1 and EWSR1/FLI1-T79D expressing cells (Dox+) and controls (Dox untreated) were subjected to a metaphase chromosome spread protocol ( Fig. 5 A ). A , Representative images of chromosomes from induced cells expressing EWSR1/FLI1 or EWSR1/FLI1-T79D and uninduced cells. (EWSR1/FLI1 Dox-; 46 chromosomes, EWSR1/FLI1 Dox+; 23 chromosomes, EWSR1/FLI1-T79D Dox-; 46 chromosomes, EWSR1/FLI1-T79D Dox+; 30 chromosomes). The color of the chromosome images was inverted using the “Invert” function of Photoshop software. The background intensity was adjusted using the “Brightness” and “Linear-Curves” functions of Photoshop. Scale bar = 10um. B , The percentage of cells that displayed aberrant numbers of chromosomes was increased in cells expressing EWSR1/FLI1-T79D, with no significant difference in the incidence of nondiploid chromosome numbers between cells expressing EWSR1/FLI1 and cells expressing EWSR1/FLI1-T79D . (20 cells per sample, n = 3 experiments). ∗∗ p < 0.01, NS: not significant (ANOVA one way with post-hoc Tukey HSD). C , The percentage of cells per sample group containing each chromosome number over the full range of chromosome numbers observed in all cells.

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

doi: 10.1074/jbc.RA120.014328

Figure Lengend Snippet: Schematic model of the study . The phosphorylation of EWSR1/FLI1 leads to the localization of EWSR1/FLI1 on mitotic chromosomes during early mitosis, with impaired localization of Aurora B at the midzone and induction of aneuploidy after a single cell cycle. The result suggests that the phosphorylation of amino acid Thr 79 of EWSR1/FLI1 has critical activity in mediating this pathway.

Article Snippet: Cell lysates from both Dox-treated and untreated cells were subjected to western blotting using a 1:1000 dilution of rabbit anti-human FLI1 (Thermo Scientific, #PA1-21023), or 1:2500 dilution of mouse anti-β-tubulin (Sigma-Aldrich, T4026) as primary antibodies, followed by treatment with a 1:100000 dilution of IRDye 680RD donkey anti-mouse IgG secondary antibody (LI-COR, #926–68072) and IRDye 680RD donkey anti-Rabbit IgG (LI-COR, #926–68073).

Techniques: Phospho-proteomics, Activity Assay