Journal: Antibodies

Article Title: A Novel FLI1 Monoclonal Antibody Which Recognizes EWS::FLI1 with High Affinity Is Useful for Detecting Ewing Sarcoma

doi: 10.3390/antib14040097

Figure Lengend Snippet: Hybridoma screening leads to novel antibody binding to EWS::FLI1. ( a ) Epitope locations on the FLI1 aligned with EWS::FLI1 ( top ). Using a sequence algorithm for predicting antigenicity we selected four 12-residue fragments to generate monoclonal antibodies. The amino acid numbers correspond to the full length FLI1 ( middle ). Likely antigenicity epitope score for each peptide was shown on the graph ( bottom ). ( b ) Immunoblot analysis showing selected custom FLI1 monoclonal antibody (1:1000 dilution) detection of EWS::FLI1 fusion protein (15 μg of total protein) in the ES cell line TC32 (type 1) and A4573 (type 3), purified recombinant EWS::FLI1 fusion protein (300 ng), and HEK293 (lack EWS::FLI1). Actin used as a loading control. ( c ) Dot blot analysis showing titration of selected FLI1 monoclonal antibody (1:1000 dilution) used against different concentration of nuclear lysates (NL) from A4573 and TC32. ( d ) Nuclear co-immunoprecipitation (co-IP) of EWS::FLI1 screening using selected FLI1 antibody clones. Immunoprecipitated RHA is shown. An amount of 10% of total nuclear lysate was used as input. Immunoblot detection of RHA and EWS::FLI1 (Abcam, Waltham, MA, USA, Cat. No. ab15289, 1:1000 dilution).

Article Snippet: A mouse monoclonal antibody (clone 3C16–FLI1 1.2) was produced (Abmart, Inc., Shanghai, China) by immunization of 6 to 8-week-old female BALB/c mice with a synthetic antigenic peptide containing amino acid residues corresponding to the C-terminal end after the DNA-binding domain (AA 376 to 387-SSMYKYPSDISY) of FLI1 ( a).

Techniques: Binding Assay, Sequencing, Residue, Bioprocessing, Western Blot, Purification, Recombinant, Control, Dot Blot, Titration, Concentration Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Clone Assay

Journal: Antibodies

Article Title: A Novel FLI1 Monoclonal Antibody Which Recognizes EWS::FLI1 with High Affinity Is Useful for Detecting Ewing Sarcoma

doi: 10.3390/antib14040097

Figure Lengend Snippet: Surface plasmon resonance (SPR) supports antibody specificity using peptide competition. For ( a – c ), antibody FLI1 1.2 was immobilized on the surface of an SPR chip. ( a ) Peptide-1, used to generate clones 1.1–1.6 binding to FLI1 1.2. ( b ) Peptide-4, used to generate clones 4.1–4.7 binding to FLI1 1.2. ( c ) Recombinant EWS::FLI1 binding to antibody FLI1 1.2. For ( a – c ), colored lines are incremental titration data and black lines are fit to the 1:1 kinetics binding model. The values are mean ± s.d. from three independent experiments. ( d ) Competition assay showing the SPR sensorgram for 100 nM EWS::FLI1 binding to immobilized FLI1 1.2 monoclonal antibody in the absence (blue) and presence of either peptide-1 (red) or -4 (yellow). When EWS::FLI1 binding was tested in the presence of peptides, the peptides were injected into the antibody immobilized surface prior to injecting EWS::FLI1. Each concentration of all samples was injected in duplicates for technical reproducibility. ( e ) Quantified EWS::FLI1 binding competition from ( d ) based on 100% binding in the absence of any peptides calculated using response values at the end of analyte injections (60 s). The values are mean ± s.d. from three independent experiments. * p < 0.05.

Article Snippet: A mouse monoclonal antibody (clone 3C16–FLI1 1.2) was produced (Abmart, Inc., Shanghai, China) by immunization of 6 to 8-week-old female BALB/c mice with a synthetic antigenic peptide containing amino acid residues corresponding to the C-terminal end after the DNA-binding domain (AA 376 to 387-SSMYKYPSDISY) of FLI1 ( a).

Techniques: SPR Assay, Clone Assay, Binding Assay, Recombinant, Titration, Competitive Binding Assay, Injection, Concentration Assay

Journal: Antibodies

Article Title: A Novel FLI1 Monoclonal Antibody Which Recognizes EWS::FLI1 with High Affinity Is Useful for Detecting Ewing Sarcoma

doi: 10.3390/antib14040097

Figure Lengend Snippet: Novel FLI1 monoclonal antibody recognizes EWS::FLI1, FLI1, and ERG. ( a ) Immunoblot analysis showing FLI1 1.2 monoclonal antibody (1:1000 dilution) detection of EWS::FLI1 fusion protein (15 μg of total protein) in various ES cell lines with EWS::FLI1 fusion type 1 (TC32, TC71, A673 and CHLA-10), type 2 (STA.ET.7.2, SKES), and type 3 (A4573). CAPAN-1 lacks EWS::FLI1. ( b ) Immunoblot analysis showing ETS proteins transfected into COS7 cells and detected using anti-FLAG antibody (top) or FLI1 1.2 monoclonal antibody (middle). EWS::FLI1 was transfected without a FLAG tag. ( c ) Competition of immunoblot detection using FLI1 1.2 in the ES cell line TC32 or A4573. HEK293 lack EWS::FLI1. FLI1 1.2 was mixed with 100 nM peptide-1 (middle) or peptide-4 (right). ( d ) Competition of immunohistochemical analysis of cell lines A4573 and TC32 using antibody FLI1 1.2. Titrated competitive inhibitor peptide-1 was added with antibody. Images are taken at 60× on Nikon SoRa Spinning Disk Microscope with the scale bar of 10 μm. ( e ) Quantification obtained after imageJ2 analysis of A4573 representative images of 5 cells for each condition. Each dot represents the value of one cell tested (* p < 0.05). ( f ) Reduction of EWS::FLI1 with IPTG induced shRNA. Cells were stained with FLI1 1.2 showing the EWS::FLI1 (green) immunofluorescence expression in the presence or reduction of EWS::FLI1 in the IPTG-inducible TC32 cells. Corresponding immunoblots showing the EWS::FLI1 expression levels on the right. For all immunoblots, beta-actin was used as a loading control.

Article Snippet: A mouse monoclonal antibody (clone 3C16–FLI1 1.2) was produced (Abmart, Inc., Shanghai, China) by immunization of 6 to 8-week-old female BALB/c mice with a synthetic antigenic peptide containing amino acid residues corresponding to the C-terminal end after the DNA-binding domain (AA 376 to 387-SSMYKYPSDISY) of FLI1 ( a).

Techniques: Western Blot, Transfection, FLAG-tag, Immunohistochemical staining, Microscopy, shRNA, Staining, Immunofluorescence, Expressing, Control

Journal: Cancers

Article Title: The Transcription Factor FEZF1 , a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes

doi: 10.3390/cancers13225668

Figure Lengend Snippet: EWSR1-FLI1 regulates the expression of a significant number of transcription factors/regulators. ( A ) A673/TR/shEF cells were cultured in the absence or presence of doxycycline (DOX, 1 µg/mL, 72 h) and analyzed by RNAseq to identify EWSR1-FLI1 regulated genes. EWSR1-FLI1 regulated genes were cross-referenced with a list of transcriptional regulators obtained from the gene ontology term “DNA-binding transcription factor activity” to identify the transcriptional regulators whose expression is downregulated or upregulated upon EWSR1-FLI1 knockdown. ( B ) Distribution of 1635 transcription factors according to its expression level (Log 2 fold change). The table shows the Log 2 fold change and the adjusted p -value from the top-fifteen transcriptional regulators that were downregulated upon EWSR1-FLI1 knockdown.

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody from Abcam (#ab133485) (Cambridge, UK), anti-FEZF1 mouse monoclonal antibody from Santa Cruz Biotechnology (#sc-515487) (Dallas, TX, USA), and HRP-anti-α-tubulin antibody from Abcam (#ab185067) (St. Louis, MO, USA).

Techniques: Expressing, Cell Culture, Binding Assay, Activity Assay

Journal: Cancers

Article Title: The Transcription Factor FEZF1 , a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes

doi: 10.3390/cancers13225668

Figure Lengend Snippet: FEZF1 expression correlates positively with EWSR1-FLI1 levels and is highly expressed in Ewing sarcoma cell lines. ( A ) FEZF1 mRNA expression (RT-qPCR) in A673/TR/shEF cells stimulated with doxycycline (DOX, 1 µg/mL, 72 h). FEZF1 mRNA levels decreased more than 80% upon EWSR1-FLI1 knockdown (mean ± SD) ( B ) Western-blot analysis of FEZF1 and EWSR1-FLI1 in A673/TR/shEF cells confirm downregulation of FEZF1 protein upon EWSR1-FLI1 knock-down (asterisk denotes an unspecific band). ( C ) A673/TR/shEF cells were cultured in the absence or presence of doxycycline and FEZF1 protein detected by immunofluorescence. FEZF1 is located in the nucleus in control cells (−DOX), but its expression is lost when cells are stimulated with doxycycline to downregulate EWSR1-FLI1 levels. Scale bar: 10 µm. ( D ) FEZF1 mRNA expression levels (RT-qPCR) in a panel of bone sarcoma cell lines including Ewing sarcoma (ES), chondrosarcoma (CS), and osteosarcoma (OS). FEZF1 was exclusively expressed in Ewing sarcoma cells. FEZF1 expression in A673/TR/shEF cells is included for comparative purposes (mean ± SD). ( E ) Western-blot analysis of FEZF1 and tubulin (TUB) in the same cell lines confirmed the expression of FEZF1 only in Ewing sarcoma cell lines. ( F ) FEZF1 mRNA levels (RT-qPCR) were analyzed using a commercial source of human RNA from normal tissues. FEZF1 is expressed specifically in the testis and brain, although even in these tissues, its expression is significantly lower than the observed in the Ewing sarcoma cell line A673 (mean ± SD). (*** p < 0.001, **** p < 0.0001; Student’s t -test).

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody from Abcam (#ab133485) (Cambridge, UK), anti-FEZF1 mouse monoclonal antibody from Santa Cruz Biotechnology (#sc-515487) (Dallas, TX, USA), and HRP-anti-α-tubulin antibody from Abcam (#ab185067) (St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Immunofluorescence

Journal: Cancers

Article Title: The Transcription Factor FEZF1 , a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes

doi: 10.3390/cancers13225668

Figure Lengend Snippet: EWSR1-FLI1 binds a GGAA-microsatellite located into the FEZF1 gene, and the number of GGAA-repeats correlates with FEZF1 expression levels. ( A ) The genomic region corresponding to FEZF1 and FEZF1-AS1 genes is shown (UCSC genome browser). The location of GGAA-microsatellite and its corresponding sequence is shown at the bottom of the figure. Publicly available ChIP-seq data demonstrate the binding of EWSR1-FLI1 to GGAA-microsatellite in two different Ewing sarcoma cell lines (A673 and SK-N-MC). H3K27ac ChIP-seq marks indicate that this site is transcriptionally active when EWSR1-FLI1 is bound to the GGAA-microsatellite, but not when EWSR1-FLI1 is knocked-down. ( B ) The length of GGAA-microsatellite was determined by PCR and fluorescent fragment analysis. Mean allele length correlates with FEZF1 mRNA expression levels (for this analysis, CADO-ES1 was excluded). ( C ) Correlation of FEZF1 and FEZF1-AS1 mRNA expression levels in Ewing sarcoma cells.

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody from Abcam (#ab133485) (Cambridge, UK), anti-FEZF1 mouse monoclonal antibody from Santa Cruz Biotechnology (#sc-515487) (Dallas, TX, USA), and HRP-anti-α-tubulin antibody from Abcam (#ab185067) (St. Louis, MO, USA).

Techniques: Expressing, Sequencing, ChIP-sequencing, Binding Assay

Journal: Cancers

Article Title: The Transcription Factor FEZF1 , a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes

doi: 10.3390/cancers13225668

Figure Lengend Snippet: FEZF1 knockdown impairs Ewing sarcoma cell proliferation. ( A ) mRNA expression levels of FEZF1, FEZF1-AS1 and EWSR1-FLI1 in three Ewing sarcoma cell lines expressing a doxycycline-inducible shRNA directed against FEZF1 mRNA. Cells were cultured for 72 h in the absence or presence of doxycycline (1 µg/mL), and mRNA expression levels were quantified by RT-qPCR (mean ± SD). ( B ) Western-blot analysis of FEZF1 and EWSR1-FLI1 in the same cell lines, confirming the downregulation of FEZF1. EWSR1-FLI1 protein levels were not affected by FEZF1 knockdown. ( C ) Cell colony formation assays show a reduction in the number of cell colonies formed upon FEZF1 knockdown. Scale bar: 10 mm. ( D ) The number of cell duplications accumulated over 30 days in which cells were cultured in the absence or presence of doxycycline. ( E ) Cells were cultured in the absence or presence of doxycycline (1 µg/mL), and DNA synthesis was analyzed by BrdU incorporation assay. FEZF1 knock-down significantly reduced DNA synthesis in the three Ewing sarcoma cell lines analyzed. For comparative purposes, the effect of EWSR1-FLI1 knock-down in A673/TR/shEF cells is also shown (mean ± SD of one experiment out of four with equivalent results; * p < 0.05, *** p < 0.001; Student’s t -test).

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody from Abcam (#ab133485) (Cambridge, UK), anti-FEZF1 mouse monoclonal antibody from Santa Cruz Biotechnology (#sc-515487) (Dallas, TX, USA), and HRP-anti-α-tubulin antibody from Abcam (#ab185067) (St. Louis, MO, USA).

Techniques: Expressing, shRNA, Cell Culture, Quantitative RT-PCR, Western Blot, DNA Synthesis, BrdU Incorporation Assay

Journal: Oncotarget

Article Title: BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma

doi: 10.18632/oncotarget.9762

Figure Lengend Snippet: ( A ) Enrichment plots show that a EWS-FLI1-activated gene signature (Riggi_Ewing_Sarcoma_Progenitor_up, 430 genes) was suppressed in A673, TC32 and TC71 cells treated with 500 nmol/L JQ1 for 24 hours. FDR: false discovery rate. NES: Normalized enrichment score. ( B ) Venn diagrams demonstrate the overlap of genes changed more than 2 folds in JQ1-treated A673 cells and a published dataset in A673 cells upon EWS-FLI1 knockdown (GEO: GSE61950). ( C ) Expression of selected EWS-FLI1 target genes in TC32 cells treated with JQ1 at indicated concentrations for 24 hours. Actin was used as the loading control. In all figures, error bars represent standard deviations with the exception of Figure . All errors were calculated from at least three technical replicates. * p < 0.05 by Student's t -test compared with control samples.

Article Snippet: Cell lysates were made using RIPA buffer (Sigma) supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (Sigma) at 4 ° C. The rabbit polyclonal antibody specific to the carboxyl-terminal region of FLI1 (sc-356) and the mouse monoclonal antibody targeting the N-terminus of EWS (sc-48404) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Expressing, Control

Journal: Oncotarget

Article Title: BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma

doi: 10.18632/oncotarget.9762

Figure Lengend Snippet: ( A ) EWS-FLI1 mRNA and ( B ) protein levels in A673, TC32 and TC71 cells treated with JQ1 at indicated concentrations for 24 hours. ( C ) EWS-FLI1 immunoblotting in Ewing sarcoma cell lines treated with 1 μmol/L JQ1 for 24 hours. *: 5838 and COG-E-352 express EWS-ERG, thus serving as a negative control for EWS-FLI1. 293T cells were transfected with pCDH-puro-EWS-FLI1 (EF) to produce recombinant EWS-FLI1 protein as the positive control. Ctrl.: vector only.

Article Snippet: Cell lysates were made using RIPA buffer (Sigma) supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (Sigma) at 4 ° C. The rabbit polyclonal antibody specific to the carboxyl-terminal region of FLI1 (sc-356) and the mouse monoclonal antibody targeting the N-terminus of EWS (sc-48404) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Negative Control, Transfection, Recombinant, Positive Control, Plasmid Preparation

Journal: Oncotarget

Article Title: BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma

doi: 10.18632/oncotarget.9762

Figure Lengend Snippet: ( A ) TC32 or ( B ) TC71 cells were treated with 500 nmol/L JQ1 for 2, 4, 8 or 24 hours. Expression of selected EWS-FLI1 target genes was shown by qRT-PCR. * p < 0.05 by Student's t -test compared with control samples.

Article Snippet: Cell lysates were made using RIPA buffer (Sigma) supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (Sigma) at 4 ° C. The rabbit polyclonal antibody specific to the carboxyl-terminal region of FLI1 (sc-356) and the mouse monoclonal antibody targeting the N-terminus of EWS (sc-48404) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma

doi: 10.18632/oncotarget.9762

Figure Lengend Snippet: ( A ) TC32 cells were infected with lentivirus directing expression of a non-targeting (NT) shRNA or shRNA selectively targeting BET genes. Expression of selected EWS-FLI1 target genes was assessed by qRT-PCR. ( B ) TC32 or ( C ) TC71 cell growth rates were calculated by normalizing cell titer readings to the mean values of the corresponding groups on day 1, which was assigned a value of 1. * p < 0.05 by unpaired Student's t -test compared with values of the NT group of the same day.

Article Snippet: Cell lysates were made using RIPA buffer (Sigma) supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (Sigma) at 4 ° C. The rabbit polyclonal antibody specific to the carboxyl-terminal region of FLI1 (sc-356) and the mouse monoclonal antibody targeting the N-terminus of EWS (sc-48404) were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Expressing, shRNA, Quantitative RT-PCR