anti fli1 mouse monoclonal antibody  (Cell Marque)

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: A schematic representation of pCTAP-Fli1. Streptavidin and calmodulin resins bind to streptavidin-binding peptide (SBP) and calmodulin-binding peptide (CBP), respectively. ATA: A-terminal activation domain, EBD: Ets binding domain, CTA: C-terminal activation domain. B. Foreskin fibroblasts were grown to subconfluence and serum-starved for 48 hours. Equal amount of protein from whole cell lysates was subjected to immunoprecipitation with or without anti-Fli1 antibody. The levels of Fli1, HDAC1, and HDAC3 proteins in precipitates were determined by immunoblotting. The levels of Fli1, HDAC1 and HDAC3 in whole cell lysates were determined by Western blotting. C. 293 T cells were transfected with pCTAP expression vector encoding wild type Fli1 along with PCAF, PCAFΔHAT, or HDAC1 expression vectors, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of Flag-tagged proteins in cell lysates were determined by Western blotting. D. 293 T cells were transfected with pCTAP wild type Fli1 along with PCAF or PCAFΔHAT expression vectors. Six hours after the transfection, HDAC1 antisense oligonucleotide (HDAC1 AS oligo) or HDAC1 sense oligonucleotide (HDAC1 S oligo) were added to the cells. Whole cells lysates were prepared 48 hours after transfection. The levels of acetylated Fli1 and Flag-tagged proteins were determined as described above. The efficacy of HDAC1 AS oligo was evaluated by Western blotting.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Binding Assay, Activation Assay, Immunoprecipitation, Western Blot, Transfection, Expressing, Plasmid Preparation, Incubation

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: 293T cells were transfected with pCTAP wild type Fli1 along with the indicated HAT proteins, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). In order to visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of HAT proteins in cell lysates were determined by Western blotting. B. Whole cell lysates prepared as described above were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-coupled agarose beads. The levels of Flag-tagged proteins and Fli1 in cell lysates were determined by Western blotting. C. 293T cells were transfected with pSG5 wild type Fli1 or Fli1 K380Q mutant along with the p300 expression vector, and incubated for 48 hours. D. 293T cells were transfected with pCTAP wild type Fli1 along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using SA beads, followed by immunoblotting using anti-HDAC1 antibody. To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of ectopically expressed p300 and endogenous HDAC1 in cell lysates were determined by Western blotting with anti-Flag antibody and anti-HDAC1 antibody, respectively.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Labeling, Isolation, Mutagenesis, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: 293T cells were transfected with pCTAP wild type Fli1 or the Fli1 T312A mutant along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads) followed by immunoblotting with anti-Flag antibody (left panels), or to immunoprecipitation using the anti-Flag antibody followed by immunoblotting with anti-calmodulin antibody (right panels). The levels of ectopically expressed p300 or Fli1 in cell lysates were determined by Western blotting with anti-Flag antibody (the bottom left panel) or anti-calmodulin antibody (the bottom right panel), respectively. B. Foreskin fibroblasts were transduced with 25 MOI of Fli1 or green fluorescence protein (GFP) adenovirus for 72 hours (left panels), or/and 25 MOI of Fli1siRNA or scrambled RNA (SCR) adenovirus for 72 hours (right panels). Nuclear extracts were subjected to immunoprecipitation with anti-p300 antibody, followed by immunoblotting with anti-HDAC1 antibody. The levels of Fli1, p300, and HDAC1 in nuclear extracts were evaluated by Western blotting. C. Whole cell lysates prepared from untreated or TGF-β stimulated foreskin fibroblasts were subjected to immunoprecipitation with anti-Fli1 antibody, followed by immunoblotting with antibodies against p300, HDAC1, or Fli1. The levels of endogenous p300 and HDAC1 were examined by Western blotting.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, Transduction, Fluorescence

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: Formaldehyde-cross-linked, moderately sheared chromatin was prepared from confluent quiescent fibroblasts treated with Trichostatin A (200 nM), anacardic acid (50 µM), or DMSO for 24 hours. The DNA fragments were immunoprecipitated using anti-acetylated histone H3 antibody, and the presence of the human α2(I) collagen (COL1A2) promoter fragments was detected using PCR. B. Formaldehyde-cross-linked, moderately sheared chromatin was prepared from foreskin fibroblasts untreated or treated with TGF-β1 (2.5 ng/ml) for 3 hours. The DNA fragments were immunoprecipitated using antibodies against Fli1, Ets1, p300, HDAC1, and acetylated histone H3. The presence of COL1A2 promoter fragments was detected using PCR.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: Foreskin fibroblasts were transfected with the −772 COL1A2/CAT construct (2 µg), along with wild type Fli1 or the Fli1 K380R mutant (0.1 µg) and HDAC1 (0, 0.1, 0.3, or 0.5 µg) for 48 h. Values represent CAT activities relative to those of untreated cells (100 arbitrary units [AU]). The mean and SD from three separate experiments are shown. * p<0.05 versus control cells. ** p<0.05 versus cells transfected with wild type Fli1 only. # p<0.05 versus control cells. ## p<0.05 versus cells transfected with Fli1 K380R only. B. Confluent quiescent foreskin fibroblasts were treated with HDAC1 inhibitor or vehicle for 24 hours. Type I procollagen protein levels in whole cell lysates were determined by immunoblotting. A representative result of three independent experiments is shown. The band density was evaluated by densitometry. C. Under the same conditions, mRNA levels of the α1(I) collagen (COL1A1) gene were determined using reverse transcription quantitative real-time PCR. The graph represents -fold change in COL1A1 mRNA levels in comparison to unstimulated controls, which were arbitrarily set at 100. The mean and SD from three separate experiments are shown. * p<0.05 versus control cells treated with vehicle.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Transfection, Construct, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

doi: 10.1371/journal.pone.0074930

Figure Lengend Snippet: In quiescent cells, Fli1 forms a transcription repressor complex with p300 and HDAC1. HDAC1/p300 promotes deacetylation of Fli1, resulting in increased Fli1 DNA binding and a repressed chromatin state. After TGF-β stimulation, Fli1 phosphorylation by protein kinase C-δ induces disassembly of this transcription repressor complex and the acetylation of Fli1 by PCAF, leading to the loss of Fli1 DNA binding. Subsequently, the transcription activator complex consisting of Ets1 and p300 binds to the Ets binding site and activates transcription at least partially by promoting histone acetylation.

Article Snippet: The monoclonal mouse anti-Fli1 antibody was purchased from BD Bioscience (Bedford, MA, USA).

Techniques: Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: EWS-FLI1 drives expression of WRN, an activity which is blocked by trabectedin treatment. A, Mean (+/− SEM) fold change in WRN expression as a function of GAPDH (2ddCT) as measured by qPCR for treatment with solvent control (S)(1.2 +/− 0.1), 10 nM(0.03 +/− 0.01), 7.5 nM (0.08 +/− 0.007) or 5 nM trabectedin ((0.23 +/− 0.02) for 12 hours P < 0.0001. Data representative of 3 independent experiments. B, Western blot time course experiment showing expression of WRN as a function of trabectedin treatment over time (hours) relative to NR0B1, ID2, PARP cleavage and ACTB (loading control). C, Western blot showing expression of WRN as a function of 10 nM trabectedin for 18 hours (ET) or solvent control (SC) in 7 different ES cell lines. Western blots representative of 3 independent experiments (time course) or 2–3 different experiments in 7 cell lines (cell line panel). D, Western blot showing expression of EWS-FLI1, WRN, the EWS-FLI1 target gene ID2, or other DNA damage genes XRCC4, ATM, KU80 and PRKDC for the medium control (C), lipid control (L) or siRNA silencing with a non-targeting siRNA (−) or siRNA targeting EWS-FLI1 (siEF) or SP1 (siSP1) in TC32 ES cellsor two other ES cell lines TC71 or EW8. Data representative of 2 (EW8) or 3 independent experiments (TC32 and TC71).

Article Snippet: Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: Trabectedin sensitizes TC32 and TC71 ES cells to SN38 treatment. A, Dose response curves of cell number at 48 hours of treatment as a function of log concentration SN38 (nM). The curves represent SN38 (circles and solid black) + 0.25 nM trabectedin (Trabec., squares and dotted dark gray) or 0.5 nM trabectedin (Trabec, triangles and solid dark gray). B, Data normalized to solvent control showing the shift in the IC50 of SN38 (black) in TC32 or TC71 cell lines with the addition of 0.5 nM trabectedin (Trabec., gray). C, Identical experiment as (B) showing shift in the IC50 of doxorubicin (black) with 0.5 nM trabectedin (Trabec., Gray). D, Dose response curves of SN38 +/− trabectedin in HT1080 cells transduced with GFP or EWS-FLI1: GFP-SN38 (black solid), GFP-SN38 + trabectedin (Trabec., gray dotted), EWS-FLI1 SN38 (black dotted) EWS-FLI1 SN38 + trabectedin (Trabec., gray solid). The curves are the mean effect determined by non-linear regression for 12 replicates from 3 to 6 independent experiments.

Article Snippet: Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques: Concentration Assay, Transduction

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: Trabectedin and SN38 cooperate to increase DNA double strand breaks and augment the suppression of EWS-FLI1 downstream targets relative to either agent alone. A, Single cell imaging showing DNA double strand breaks as measured by the formation and immunostaining of γH2AX foci (green) with DAPI stained nuclei for reference (blue) following treatment of TC32 cells with 5 nM trabectedin, SN38 or the combination for 6 hours. B, Populations of TC32 cells showing the increased penetrance of γH2AX in the combination therapy relative to each agent alone. White arrows highlight unaffected cells. Quantitation of γH2AX foci in 50 to 65 cells from 8 different images in each of the treatment groups, Control (C), Trabectedin (T), SN38 (SN) or the combination (T&SN). C, Normalized bioluminescence of stable NR0B1 promoter construct (EWS-FLI1 reporter) treated with solvent control (S) or the designated concentration of trabectedin (Trabec) or SN38 (* & ^ denote comparisons for P< 0.0005). Data represents the mean value normalized to the solvent control for 8 replicates from two independent experiments for each treatment group. D, Western blot showing the effect of SN38 treatment (concentrations above the blot) +/− 5 or 1 nM trabectedin (T) on PARP cleavage, EWS-FLI1 downstream target expression (ID2) and γH2AX expression. Identical results obtained with three independent experiments. (*) Highlights specific concentrations of trabectedin, SN38 and the combination for comparison.

Article Snippet: Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques: Imaging, Immunostaining, Staining, Quantitation Assay, Construct, Concentration Assay, Western Blot, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

doi: 10.1158/1078-0432.CCR-13-0901

Figure Lengend Snippet: Recapitulation of the WRN null phenotype. A, siRNA mediated silencing of WRN (siWRN) for 48 hours sensitizes TC32 Ewing sarcoma cells to treatment with 1 nM SN38. The controls are a non-targeting siRNA (siNEG) and a siRNA targeting EWS-FLI1 (siEWS-FLI1). B, Silencing of WRN for 48 hours sensitizes TC71 Ewing sarcoma cells to treatment with 0.5 nM SN38. The controls are a non-targeting siRNA (siNEG) and a siRNA targeting EWS-FLI1 (siEWS-FLI1). C, Cell cycle effects of ET743 and SN38 that recapitulate the WRN null phenotype. Treatment of TC32 Ewing sarcoma cells with 1 nM Trabectedin and SN38 for 18 hours shifts the cell cycle to S & G2M as shown in these representative histogram plots. D, Column statistics for the treatment groups for the subG0, G1, S and G2 gates. Treatment groups are untreated (medium), solvent control (solvent), 1 nM Trabectedin (Trabectedin) and 1 nM SN38 (SN38). Data represents the mean (+/− St Dev) from at least two independent experiments. P values represent the statistical significance of the changes in S-phase relative to the solvent control and the comparisons are denoted with + or ++.

Article Snippet: Immunoblotting was performed as previously described ( 39 ) using the following antibodies: mouse monoclonal anti-FLI1 (1.5 μg/mL; Becton Dickinson (BD), Franklin Lakes, NJ), anti-WRN (1:1000, Cell Signaling, Danvers, MA), anti-ATM (1:1000, Cell Signaling), anti-XRCC4 (1:1000, Abcam, Cambridge, UK), rabbit monoclonal anti-ID2 (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-NR0B1 (1:1000 dilution; Abcam), mouse monoclonal anti-PARP (1:1000 dilution, Cell Signaling), rabbit polyclonal anti-ACTB (1:10,000 dilution, Abcam), mouse monoclonal anti-phospho-histone H2A.X (ser 139)(γH2AX)(1:1000 dilution, Millipore, Billerica, MA).

Techniques:

Journal: Scientific Reports

Article Title: 18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

doi: 10.1038/srep33926

Figure Lengend Snippet: (A) Schema showing theory of study. Suppression of an EWS-FLI1 target expressed on the cell surface will allow quantitation of EWS-FLI1 activity based on loss of transport of a labeled substrate or loss of binding of a labeled antibody or ligand. (B) Heat map summarizing screen results. EWS-FLI1 was silenced in three different cell lines (TC32, TC71 and EW8) and the induction (red) or suppression (blue) of expression was measured by qPCR. ENT1, ENT2, and TK1 (arrows) were all suppressed with silencing of EWS-FLI1. (C) Confirmatory PCR in 4 cell lines showing suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1 as measured by qPCR. ( P for all treatments vs. no change) (D) Western blots confirm suppression of ENT1, ENT2, and TK1 with siRNA silencing of EWS-FLI1.

Article Snippet: The membrane was probed with the following antibodies: mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling, Danvers, MA), rabbit polyclonal anti-ENT1 (1:500, Abcam), rabbit polyclonal anti-ENT2 (1:1000, Abcam), and rabbit polyclonal anti-TK1 (1:000, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Quantitation Assay, Activity Assay, Labeling, Binding Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: 18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

doi: 10.1038/srep33926

Figure Lengend Snippet: ( A ) Treatment of TC32 Ewing sarcoma cells with 50 nM mithramycin (MMA) phenocopied the suppression of ENT1, ENT2, and TK1 expression that occurs with siRNA silencing of EWS-FLI1. ( B ) Treatment of TC32 Ewing sarcoma cells with the mithramycin analogs EC-8042 and EC-8105 suppressed ENT1, ENT2, and TK1, but 5-FU induced their expression ( P for all treatments vs. no change) ( C ) Expression of ENT1, ENT2, and TK1 was unchanged or induced in 4 control cell lines that do not express EWS-FLI1 with treatment with mithramycin or 5-FU.

Article Snippet: The membrane was probed with the following antibodies: mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling, Danvers, MA), rabbit polyclonal anti-ENT1 (1:500, Abcam), rabbit polyclonal anti-ENT2 (1:1000, Abcam), and rabbit polyclonal anti-TK1 (1:000, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing

Journal: Scientific Reports

Article Title: 18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

doi: 10.1038/srep33926

Figure Lengend Snippet: ( A ) 18 F-FLT PET activity decreased in vitro in TC32 Ewing sarcoma cells with siRNA silencing of EWS-FLI1(siEWS) but not with a non-targeting control (siNeg); EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105 decreases 18 F-FLT activity ( B ) 18 F-FDG activity does not change with siRNA silencing of EWS-FLI1 and with EWS-FLI1 suppression with mithramycin (MMA), EC-8042, or EC-8105. 5FU suppresses 18 F-FDG activity likely due to a loss of cell viability.

Article Snippet: The membrane was probed with the following antibodies: mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling, Danvers, MA), rabbit polyclonal anti-ENT1 (1:500, Abcam), rabbit polyclonal anti-ENT2 (1:1000, Abcam), and rabbit polyclonal anti-TK1 (1:000, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Activity Assay, In Vitro

Journal: Scientific Reports

Article Title: 18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

doi: 10.1038/srep33926

Figure Lengend Snippet: The suppression of 18 F-FLT activity only occurs with EWS-FLI1 suppression and can not be explained by non-specific changes in cell cycle progression ( A ) Cell cycle changes in TC32 Ewing sarcoma cells that occur with siRNA silencing of EWS-FLI1 or treatment with 50 nM mithramycin (MMA) or the mithramycin analogs EC8105 (15 nM) or EC8042 (50 nM). ( B ) Cell counts as percentage of total (+/− SD). Grey boxes highlight treatments that increase the number of cells in G1 or S. Treatments identical to ( C ) Treatment of TC32 Ewing sarcoma cells with non-specific chemotherapy: 2 nM vincristine (VCR), 80 nM etoposide (etop) or 50 nM doxorubicin (doxo) does not suppress 18 F-FLT activity in comparison to mithramycin (MMA). Cells treated equitoxic concentrations.

Article Snippet: The membrane was probed with the following antibodies: mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling, Danvers, MA), rabbit polyclonal anti-ENT1 (1:500, Abcam), rabbit polyclonal anti-ENT2 (1:1000, Abcam), and rabbit polyclonal anti-TK1 (1:000, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification of mithramycin analogs with improved targeting of the EWS-FLI1 transcription factor

doi: 10.1158/1078-0432.CCR-15-2624

Figure Lengend Snippet: A, IC50 of suppression of EWS-FLI1 activity for a panel of mithramycin (MMA, black arrow) and mithramycin analogs including EC-8105 (grey arrow); graphs separated due to disparity in scale. B, Dose–response curve and corresponding IC50 of suppression of EWS-FLI1 activity as determined by nonlinear regression for EC-8105 relative to EC-8041 (non-DNA binding) and mithramycin (MMA). C, Mean (±SEM) fold change in NR0B1 expression as a function of GAPDH (2ΔΔCT) for a 12 hr treatment with solvent control (S) or compound. D, Cell viability IC50 (nmol/L) for EC-8105 and mithramycin at 48 h of treatment as determined by nonlinear regression from 3 independent experiments. E, Heat map of ΔΔCt score as a measure of induction (red) or repression (blue) of expression for EWS-FLI1-induced (top) and repressed (bottom) targets as a function of GAPDH for EC-8105 treatment at 15 nM for 18 h or 3 h, respectively. F, Western blots from TC32 and TC71 cell lines showing the effect of mithramycin or EC-8105 treatment for 18 hours at the indicated concentrations (nmol/L) on EWS/FLI1 and downstream target expression (EZH2, NR0B1, and ID2), with ACTB as a loading control. All qPCR data is the average of 3 independent experiments and all other results are representative of 3 independent experiments.

Article Snippet: Thirty micrograms of protein was resolved on a 4–12% NuPAGE Bis-Tris Mini gels (Invitrogen, Carlsbad, CA) in 1x 4-morpholinepropanesulfonic acid (MOPS) sodium dodecyl sulfate (SDS) buffer (Invitrogen), transferred to nitrocellulose (GE Healthcare Life Sciences, Pittsburgh, PA) and probed with the following antibodies: rabbit monoclonal anti-EZH2 (1:1,000, Cell Signaling, Danvers, MA), mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling), rabbit polyclonal anti-NR0B1 (1:500, Abcam), mouse monoclonal anti-phospho-histone H2A.X (Ser139) (1:1,0000, Millipore, Billerica, MA), and rabbit monoclonal anti-ID2 (1:1,000, Cell Signaling).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification of mithramycin analogs with improved targeting of the EWS-FLI1 transcription factor

doi: 10.1158/1078-0432.CCR-15-2624

Figure Lengend Snippet: A, Mean (±SEM) fold change in NR0B1 expression as measured by qPCR as a function of GAPDH (2ΔΔCT) for treatment for 18 h with solvent control (S) or drugs as shown. B, Heat map of ΔΔCt score as a measure of induction (red) or repression (blue) of expression for EWS-FLI1-induced (top) and repressed (bottom) targets as a function of GAPDH for EC-8042 treatment at 15 nM for 18 h or 3 h, respectively C, Heat map of ΔΔCt score as a measure of induction (red) or repression (blue) of expression for EWS-FLI1 gene signature in control cell lines (U2OS, osteosarcoma, RD, embryonal rhabdomyosarcoma and RH30 alveolar rhabdomyosarcoma) following treatment for 18 hours with 50 nmol/L mithramycin (MMA), 50 nmol/L EC-8042 or 15 nmol/L EC-8105 for 18 h. D, Immunoblot from TC32 and TC71 cell lines showing the effect of mithramycin or EC-8042 treatment on EWS-FLI1 downstream target expression (EZH2, NR0B1, and ID2) and γH2AX phosphorylation with ACTB as a loading control. E, Dose response curves of cell number at 48 h of treatment. All qPCR data is the average of 3 independent experiments and all other results are representative of 3 independent experiments.

Article Snippet: Thirty micrograms of protein was resolved on a 4–12% NuPAGE Bis-Tris Mini gels (Invitrogen, Carlsbad, CA) in 1x 4-morpholinepropanesulfonic acid (MOPS) sodium dodecyl sulfate (SDS) buffer (Invitrogen), transferred to nitrocellulose (GE Healthcare Life Sciences, Pittsburgh, PA) and probed with the following antibodies: rabbit monoclonal anti-EZH2 (1:1,000, Cell Signaling, Danvers, MA), mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling), rabbit polyclonal anti-NR0B1 (1:500, Abcam), mouse monoclonal anti-phospho-histone H2A.X (Ser139) (1:1,0000, Millipore, Billerica, MA), and rabbit monoclonal anti-ID2 (1:1,000, Cell Signaling).

Techniques: Expressing, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification of mithramycin analogs with improved targeting of the EWS-FLI1 transcription factor

doi: 10.1158/1078-0432.CCR-15-2624

Figure Lengend Snippet: A, Mean fold change in expression of EWS-FLI1-induced targets or -repressed targets as a function of GAPDH (2ΔΔCT) after treatment with 50 nmol/L mithramycin, 50 nmol/L EC-8042 or 7.5 nmol/L EC-8105 for 12 h. qPCR data is the average of 3 independent experiments. B, Time-lapse microscopy demonstrating suppression of TC32 Ewing sarcoma cell proliferation over time following drug addition and EWS-FLI1 target suppression (black arrow) for 50 nmol/L mithramycin, 50 nmol/L EC-8042 or 7.5 nmol/L EC-8105 C, End point MTS assay confirming effect on cell viability in TC32 cells. D, Time-lapse microscopy demonstrating suppression of HepG2 proliferation with 50 nmol/L mithramycin but not 50 nmol/L EC-8042 or 7.5 nmol/L EC-8105. E, End point MTS assay confirming effect on cell viability in HepG2 cells. F, Confocal microscopy and G, western blot analysis demonstrates induction of DNA damage as measured by the phosphorylation of γH2AX at concentrations that suppress expression of the EWS-FLI1 target gene NR0B1 only with 50 nmol/L mithramycin and not with 50 nmol/L EC-8042 or marginally with 7.5 nmol/L EC-8105.

Article Snippet: Thirty micrograms of protein was resolved on a 4–12% NuPAGE Bis-Tris Mini gels (Invitrogen, Carlsbad, CA) in 1x 4-morpholinepropanesulfonic acid (MOPS) sodium dodecyl sulfate (SDS) buffer (Invitrogen), transferred to nitrocellulose (GE Healthcare Life Sciences, Pittsburgh, PA) and probed with the following antibodies: rabbit monoclonal anti-EZH2 (1:1,000, Cell Signaling, Danvers, MA), mouse monoclonal anti-FLI1 (1:1,000, Abcam, Cambridge, MA), mouse polyclonal anti-ACTB (1:1,000, Cell Signaling), rabbit polyclonal anti-NR0B1 (1:500, Abcam), mouse monoclonal anti-phospho-histone H2A.X (Ser139) (1:1,0000, Millipore, Billerica, MA), and rabbit monoclonal anti-ID2 (1:1,000, Cell Signaling).

Techniques: Expressing, Time-lapse Microscopy, MTS Assay, Confocal Microscopy, Western Blot