Journal: Journal of Biological Chemistry

Article Title: Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation

doi: 10.1074/jbc.m503288200

Figure Lengend Snippet: FIG. 3. Treatment of LS8 cells with NaF induces the ER stress response. A, representative Northern blots with 10 g total RNA/lane. Treatment times and doses are indicated above each lane, and the identity of the mRNA assayed is listed beside each blot. The same blot was stripped and reprobed for each mRNA assayed. Treatment of LS8 cells with tunicamycin (Tun) served as the ER stress response positive control (cont). With the exception of the -actin control, the stress response genes assayed displayed increased expression in a time- and dose-dependent manner after NaF treatment. B, Northern blot analysis of LS8 gene expression as a function of pretreatment with z-VAD (Z). The same filter was stripped and reprobed for each mRNA assayed. Tunicamycin (T) served as the positive control. Note that the absence of DNA fragmentation did not affect NaF-mediated gene transcription. C, Western blot of protein (30 g) isolated from LS8 cell nuclei after 24 h of treatment with NaF (2 mM) or tunicamycin (0.5 g/ml). Both GADD153 and the active form of Xbp1 were induced by each treatment. D, immunohistochemical staining of LS8 cell nuclei with a monoclonal antibody specific for GADD153. Cells were treated or not with 2 mM NaF for 24 h as indicated. Panels to the left were stained with both primary and secondary antibodies (Ab). Panels to the right were stained with secondary antisera only. Immunolabeling detected strong expres- sion of GADD153 in the NaF-treated cells.

Article Snippet: Antibodies (Santa Cruz Biotechnologies) were specific for GADD153 (sc-7351, 1:2000) or Xbp-1 (M-186, 1:3000).

Techniques: Northern Blot, Positive Control, Control, Expressing, Gene Expression, Western Blot, Isolation, Immunohistochemical staining, Staining, Immunolabeling

Journal: Journal of Biological Chemistry

Article Title: Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation

doi: 10.1074/jbc.m503288200

Figure Lengend Snippet: FIG. 5. Attenuation of GADD153 expression increases NaF-in- duced DNA fragmentation in LS8 cells, whereas transient over- expression has little effect. A, representative Western blots of GADD153 expression. Top, pretreatment with GADD153 siRNA (NaF, ) reduced the NaF-induced GADD153 up-regulation (NaF, ) to levels approximately equivalent to control cultures pretreated with nonspe- cific siRNA-only (cont, ). Each lane contains 30 g nuclear protein (top). Transient transfection of either 0.75 g (, middle lane) or 1.5 g (, right lane) CMV-GADD153 vector dramatically increased GADD153 expression over that of cells treated with empty CMV vector alone (, left lane). Each lane contains 5 g of whole cell protein (bottom). B, ELISA-based TUNEL assays for quantification of DNA strand breaks. NaF-induced DNA fragmentation of cells pretreated with GADD153 siRNA (NaF, ) demonstrated significantly more strand breaks (left panel) than those treated with nonspecific siRNA and NaF (NaF, ) or with siRNA alone (cont, ). Results similar to the NaF treatments were obtained with 0.1 g/ml tunicamycin (Tu) treat- ment for 24 h (right panel). C, ELISA-based TUNEL assay for quanti- fication of DNA strand breaks. Cells were transfected with either empty CMV vector () or CMV-GADD153 vector () and were untreated (cont) or were treated with NaF (NaF). Regardless of NaF treatment, GADD153 overexpression had little to no effect on DNA fragmentation. All NaF treatments were 5 mM for 24 h, and for all TUNEL assays, two wells of a 96-well plate were analyzed for each experimental treatment, and three separate experiments were performed. Error bars represent the S.D.

Article Snippet: Antibodies (Santa Cruz Biotechnologies) were specific for GADD153 (sc-7351, 1:2000) or Xbp-1 (M-186, 1:3000).

Techniques: Expressing, Over Expression, Western Blot, Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, TUNEL Assay

Journal: Journal of Biological Chemistry

Article Title: Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation

doi: 10.1074/jbc.m503288200

Figure Lengend Snippet: FIG. 6. Caspase-3 activity is responsible for increased levels of NaF-mediated DNA fragmentation in GADD153/ cells versus controls. Mouse embryonic fibroblasts with (GADD153/) or without (GADD153/) homozygous GADD153 gene deletion were pretreated or not with either 50 M z-VAD (top panel) or 2 M z-DEVD (bottom panel) for 1 h followed by 24 h of treatment with 5 mM NaF. Adherent cells were assayed for DNA fragmentation by the ELISA-based TUNEL assay. Three wells were assayed for each experimental treatment, and three separate experiments were performed. Error bars represent the S.E. of the mean. Note that significantly more NaF-induced strand breaks occurred in the GADD153/ cells than in the control cells and that inhibition of caspase-3 with z-DEVD eliminated this difference. Cont, control.

Article Snippet: Antibodies (Santa Cruz Biotechnologies) were specific for GADD153 (sc-7351, 1:2000) or Xbp-1 (M-186, 1:3000).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Control, Inhibition

Journal: Cell Death & Disease

Article Title: Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells

doi: 10.1038/s41419-019-1577-2

Figure Lengend Snippet: a O-GlcNAcylation was upregulated in breast cancer cells after treatment with Nic. Breast cancer cells were treated with 100 μM Nic for the indicated times. Cellular O-GlcNAcylation and the expression of EMT markers in breast cancer cell lines were analyzed by immunoblotting analysis. ( b , c ) MCF-7 and MDA-MB-231 cells were transfected with scrambled siRNA (−) or OGT siRNA (siOGT, +) for 48 h and then treated with 100 μM Nic for an additional 24 h. Protein levels were examined by immunoblotting. Transwell invasion assays showed that the reduction of O-GlcNAcylation inhibited the Nic-induced invasion of breast cancer cells. d The EMT-related proteins Snail and Vimentin were O-GlcNAcylated during Nic treatment. MCF-7 and MDA-MB-231 cells were transfected with scrambled siRNA (−) or OGT siRNA (siOGT, +) for 48 h and then treated with 100 μM Nic for 24 h. 100 μM L01 was used to inhibit O-GlcNAcylation. Snail and Vimentin immunoprecipitations assays were performed, and the immunoprecipitated fractions were analyzed by immunoblotting. e Immunocytochemistry of MCF-7 and MDA-MB-231 cells treated with 100 μM Nic either alone or together with 100 μM L01 for 12 h. The immunoreactivity of the antibodies for O-GlcNAc CTD110.6 and EMT markers is shown in green, and DAPI is shown in blue. f Wound healing assays and Transwell migration and invasion assays showed that the reduction of O-GlcNAcylation inhibited the Nic-induced migration and invasion of breast cancer cells. Cells were treated with 100 μM Nic either alone or together with 100 μM L01 for 24 h and 48 h. Graphs comparing the average migration rate in different treated cells are shown. The data represent the mean ± SEM, N = 3, * p < 0.05, ** p < 0.01

Article Snippet: OGT siRNA (sc-40780), GFAT siRNA (sc-6068), CEBPB siRNA (sc-29862), CHOP siRNA (sc-156118), XBP1 siRNA (sc-38627) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation, Immunocytochemistry, Migration

Journal: Cell Death & Disease

Article Title: Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells

doi: 10.1038/s41419-019-1577-2

Figure Lengend Snippet: a Schematic description of the putative GFAT distal promoter with potential CEBPB-binding sites. A wild-type GFAT promoter (WT) luciferase reporter construct and a mutant (MUT) form lacking the CEBPB-binding site are shown. b 293T cells were transfected with a reporter vector consisting of luciferase cDNA fused to the GFAT promoter. The pGL3 vector and mutational (MUT) GFAT promoter were used as controls. Transfection of CEBPB increased GFAT promoter activity. c MCF-7 and MDA-MB-231 cells were treated with or without 100 μM Nic for 24 h. The lysates were subjected to ChIP. Quantitative PCR amplification was performed using primers specific for the GFAT promoter. d The induction of UDP-GlcNAc by Nic was attenuated by CEBPB inhibition. Chromatograms of polar metabolites in MCF-7 and MDA-MB-231 cell extracts from cells treated with either 100 μM Nic alone (red line) or CEBPB siRNA plus Nic (blue line) for 24 h show regions corresponding to the UDP-GlcNAc derivative, UDP-GalNAc derivative and probenecid retention times. e GFAT transcriptional levels were reduced in breast cancer cells after CEBPB knockdown. The GFAT transcript level was analyzed by quantitative RT-PCR. Scrambled siRNA was used as a control. f MCF-7 and MDA-MB-231 cells were transfected with scrambled siRNA (-) or OGT siRNA (siCEBPB, +) for 48 h and then treated with 100 μM Nic for an additional 24 h. The protein levels were examined by immunoblotting. The data represent the mean ± SEM, N = 3, * p < 0.05, ** p < 0.01

Article Snippet: OGT siRNA (sc-40780), GFAT siRNA (sc-6068), CEBPB siRNA (sc-29862), CHOP siRNA (sc-156118), XBP1 siRNA (sc-38627) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Binding Assay, Luciferase, Construct, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Amplification, Inhibition, Knockdown, Quantitative RT-PCR, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells

doi: 10.1038/s41419-019-1577-2

Figure Lengend Snippet: a MCF-7 and MDA-MB-231 cells were treated with 100 μM Nic either alone or together with L01 (100 μM)/PugNAc (50 μM) for 24 h. The lysates were subjected to ChIP assays. Quantitative PCR amplification was performed using primers specific for GFAT promoter. b Indicated cells were transfected with a reporter vector consisting of luciferase cDNA fused to GFAT promotor. Transfection of OGT siRNA or inhibition of O-GlcNAc (100 μM L01 ) decreased GFAT promotor activity. PugNAc (50 μM) increased GFAT promotor activity. c O-GlcNAcylation regulates GFAT transcription in breast cancer cells with Nic treatment (100 μM, 24 h). Transfection of OGT siRNA or inhibition of O-GlcNAc (100 μM L01 ) decreased GFAT transcription. PugNAc (50 μM) increased GFAT transcription. GFAT transcript level was analyzed by quantitative RT-PCR. Scrambled siRNA was used as a control (CTRL). The data represent the mean ± SEM, N = 3, * p < 0.05, ** p < 0.01

Article Snippet: OGT siRNA (sc-40780), GFAT siRNA (sc-6068), CEBPB siRNA (sc-29862), CHOP siRNA (sc-156118), XBP1 siRNA (sc-38627) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Amplification, Transfection, Plasmid Preparation, Luciferase, Inhibition, Activity Assay, Quantitative RT-PCR, Control

Journal: Cell Death & Disease

Article Title: Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells

doi: 10.1038/s41419-019-1577-2

Figure Lengend Snippet: a CHOP/CEBPB dimer is reduced in breast cancer cells with Nic treatment (100 μM, 24 h). CHOP co-IP were performed, and immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins. b Indicated cells were transfected with a reporter vector consisting of luciferase cDNA fused to GFAT promoter and then treated for 24 h. CHOP knockdown with siRNA (siCHOP) increased GFAT promoter activity. c , d MCF-7 and MDA-MB-231 cells were transfected with scrambled siRNA (−) or CHOP siRNA (siCHOP, +) for 48 h and then treated with 100 μM Nic for an additional 24 h. Protein levels were examined by immunoblotting. Transwell invasion assays showed that reduction of O-GlcNAcylation inhibited Nic-induced invasion in breast cancer cells. e , f MDA-MB-231 cells were treated with 100 μM Nic either alone or together with transfection of pcDNA3-CHOP for 24 h. pcDNA3.1 vector was used as a control (CTRL). The lysates were subjected to ChIP assays with indicated antibodies. Quantitative PCR amplification was performed using primers specific for GFAT promoter. The protein levels were examined by immunoblotting. g O-GlcNAc modification of CHOP is an obstacle for transcriptional inhibition of CEBPB. MCF-7 and MDA-MB-231 cells were treated with 100 μM Nic either alone or together with L01 (100 μM)/PugNAc (50 μM) for 24 h. Immunoprecipitation was performed, and immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins. The data represent the mean±SEM, N = 3, * p < 0.05

Article Snippet: OGT siRNA (sc-40780), GFAT siRNA (sc-6068), CEBPB siRNA (sc-29862), CHOP siRNA (sc-156118), XBP1 siRNA (sc-38627) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Luciferase, Knockdown, Activity Assay, Control, Real-time Polymerase Chain Reaction, Amplification, Modification, Inhibition

Journal: Cell Death & Disease

Article Title: Elevation of O-GlcNAc and GFAT expression by nicotine exposure promotes epithelial‐mesenchymal transition and invasion in breast cancer cells

doi: 10.1038/s41419-019-1577-2

Figure Lengend Snippet: Nic stimulates CEBPB-dependent transcription of GFAT and then mediates the production of UDP-GlcNAc and hyper-O-GlcNAcylation. Excess O-GlcNAcylation promotes the activation of EMT and breast cancer cell invasion. The O-GlcNAcylation of CHOP also prevents its inhibition of CEBPB, subsequently enhancing GFAT transcription in a positive feedback manner

Article Snippet: OGT siRNA (sc-40780), GFAT siRNA (sc-6068), CEBPB siRNA (sc-29862), CHOP siRNA (sc-156118), XBP1 siRNA (sc-38627) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Activation Assay, Inhibition

Journal: Biomedicines

Article Title: Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response

doi: 10.3390/biomedicines8100384

Figure Lengend Snippet: Effect of idebenone on mRNA levels of endoplasmic reticulum stress (ER stress) markers in the Winnie model of spontaneous chronic colitis. Gene expression of ( A ) GRP78 , ( B ) CHOP , ( C ) ATF6 , ( D ) XBP-1 and ( E ) PERK were determined for the proximal (PC) and distal colon (DC) for all the three groups. mRNA levels were normalized to GAPDH gene and presented as fold change. Data represents mean ± SEM ( n = 4/group) using One-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The membranes were then blocked in 5% skim milk prepared in TBST and incubated overnight with primary antibody against NQO-1 (1:1000, ab34173, Abcam), GRP78 (1:1000, NBP1-06274, Novus biologicals) CHOP (1:1000, NBP2-13172, Novus Biologicals) and β-actin (1:8000, NB600-503, Novus Biologicals) at 4 °C.

Techniques: Gene Expression

Journal: Biomedicines

Article Title: Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response

doi: 10.3390/biomedicines8100384

Figure Lengend Snippet: Effect of idebenone on endoplasmic reticulum stress (ER stress) and oxidative stress in spontaneous chronic colitis in Winnie mice. ( A ) protein levels of GRP78, CHOP and NQO-1 were analyzed using western blotting of tissue samples from the distal colon (DC), ( B – D ) densitometry of GRP78, CHOP and NQO-1 expression. Band densities were normalized to β-actin. ( E ) malondialdehyde (MDA) levels in distal colon. Data represents mean ± SEM ( n = 3/group). Statistical significance was determined using One-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001 and ns: nonsignificant.

Article Snippet: The membranes were then blocked in 5% skim milk prepared in TBST and incubated overnight with primary antibody against NQO-1 (1:1000, ab34173, Abcam), GRP78 (1:1000, NBP1-06274, Novus biologicals) CHOP (1:1000, NBP2-13172, Novus Biologicals) and β-actin (1:8000, NB600-503, Novus Biologicals) at 4 °C.

Techniques: Western Blot, Expressing

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: The three normal RNA‐binding FET proteins consist of N‐terminal repetitive and structurally disordered domains (NTD: N‐terminal domain), central RNA‐binding domains (RRM: RNA recognition motifs), zinc finger domains (ZF), and degenerated repeat regions (RGG: RGG repeat regions). The GST‐tagged FET‐NTD baits shown were used for pulldown experiments and represent the shortest parts commonly present in the FET fusion oncoproteins. The FET‐binding motif is a conserved sequence required for complex formation between the three FET proteins. Amino acid numbers are indicated. Schematic illustration of a representative FET fusion protein (FUS‐DDIT3 type II) and its parental proteins. DBD: DNA‐binding domain, LZ: leucine zipper domain. Type I and type II show locations of the two most common MLS fusion breakpoints in FUS. Coomassie staining of SDS–PAGE‐separated pulldown samples with/without cell extracts (C.E) using GST‐tagged FET‐NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E −). Several high‐molecular‐weight proteins are retained by the FET‐NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry (see also Table EV1 ). Immunoblot analysis (IB) of pulldown samples with FET‐NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1, and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3, and C‐terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS‐DDIT3/BRG1, and FUS‐DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645‐94 and 402‐91. C‐terminal parts of FUS are not present in the FUS‐DDIT3 fusion protein, and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: RNA Binding Assay, Binding Assay, Sequencing, Staining, SDS Page, Control, Recombinant, High Molecular Weight, Mass Spectrometry, Western Blot, In Situ, Ligation

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Summary of SWI/SNF components as well as FET proteins (wild‐type and fusion oncoproteins) in the bound fraction after BRG1 Co‐IP of MLS 402‐91, MLS 2645‐94, EWS TC‐71, and EWS IOR/CAR, after DDIT3 Co‐IP of MLS 402‐91 and after FLI1 Co‐IP of EWS TC‐71 as verified by mass spectrometry (MS), and by immunoblot (IB) for key proteins. Note that some SWI/SNF components are represented by one of several alternative variants, for example BAF45A‐D. Lists of peptides for each mass spectrometry hit are shown in Tables EV2 and EV3

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Mass Spectrometry, Western Blot, Tandem Mass Spectroscopy

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of proteins co‐immunoprecipitated with BRG1. Detection with antibodies against BRG1, EWSR1, and FUS‐DDIT3 in MLS cell lines 402‐91 and 2645‐94. Immunoblot analysis (IB) of proteins co‐immunoprecipitated with BRG1. Detection with antibodies against and BRG1 and EWSR1 in EWS cell lines TC‐71 and IOR/CAR. CE: cytoplasmic extract, NE: nuclear extract, B FA : bound proteins eluted with 1% formic acid, B LDS : bound proteins eluted with LDS sample buffer, NB: non‐bound proteins, not captured by the antibody. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of proteins co‐immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‐91 and EWS cell line TC‐71. Antibodies against BRG1, DDIT3 (FUS‐DDIT3), the C‐terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1‐FLI1) were used for detection. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for FUS‐DDIT3, EWSR1, and FUS in MLS 402‐91 cell line and EWSR1‐FLI1, EWSR1, and FUS in EWS TC‐71 cell line. Mean ± SEM is shown with individual replicates indicated by circles ( n = 3). Student's t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001. Original data for all quantifications, including P ‐values, are shown as source data. Immunoblot analysis of proteins co‐immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS‐DDIT3‐dsRED, EWSR1‐FLI1‐EGFP, or dsRED is shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS‐DDIT3‐dsRED), the C‐terminal parts of normal FUS and EWSR1, EZH2, and FLI1 (EWSR1‐FLI1‐EGFP) were used for detection. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED‐ or EGFP‐tagged proteins expressed). Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for the fusion protein (FUS‐DDIT3 or EWSR1‐FLI1), EWSR1, FUS, and EZH2 in the model cell line HT1080 with overexpressed FUS‐DDIT3‐dsRED/‐EGFP ( n = 4, circles), EWSR1‐FLI1‐EGFP ( n = 3, squares) or dsRED/EGFP ( n = 4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t ‐test, no significant changes, P > 0.05. Original data for all quantifications, including P ‐values, are shown as source data. Four alternative models for binding of normal and oncogenic FET proteins to SWI/SNF. (I): FET oncoproteins bind directly to SWI/SNF. (II): normal FET proteins form homo‐ and hetero‐complexes and mediate binding of FET oncoproteins. (III): FET oncoproteins and normal FET proteins bind to different variants of the SWI/SNF complex with distinct biochemical compositions. (IV): FET oncoproteins and normal FET proteins bind to different sites on the SWI/SNF complex. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation, Binding Assay

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‐DDIT3‐EGFP, or EWSR1‐FLI1‐EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. More immunoblots including a second stable biological replicate and analysis of histone modifications after transient transfection (24 and 48 h) are shown in Fig A–D. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection (see Figs A, and A and F) and by squares from two experiments with transient transfection (24 and 48 h, see Fig B and G), n = 4. Student's t ‐test, ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Original data for all quantifications, including P ‐values, are shown as source data. SWI/SNF opposes the polycomb complex PRC2. The catalytic subunit of PRC2, EZH2, catalyzes the trimethylation of Lys27 on histone H3 (H3K27me3), a chromatin modification associated with closed chromatin, leading to downregulation of polycomb‐regulated genes. FET fusion binding to SWI/SNF might compromise the function of SWI/SNF, including polycomb opposition. In particular, genes downregulated when FUS‐DDIT3 is overexpressed in HT1080 cells (RNA‐seq data, n = 3, compared to control, n = 4) overlap with upregulated gene‐sets after EZH2 knockdown, NIPP1 knockdown and HDAC knockdown, and BAF57 (SMARCE1) reconstitution in a SMARCE1 null cell line. RNA‐seq data of HT1080 wt ( n = 4), HT1080 EGFP ( n = 4), HT1080 FUS‐DDIT3‐EGFP ( n = 3), and HT1080 EWSR1‐FLI1‐EGFP ( n = 4) were generated and genes > twofold regulated were analyzed using the molecular signature database (MSigDB). Gene lists were compared to the gene‐set collection “chemical and genetic perturbations” and top 20 gene‐sets for each comparison are shown in the source data. q ‐values (FDR‐adjusted P ‐values) are indicated in parentheses. Schematic presentation of FET family of fusion oncoproteins, targeted DNA sequences, and associated tumors. Left: FET N‐terminal fusion partners and binding to SWI/SNF. Center: C‐terminal transcription factor fusion partners and their DNA target sequences (JASPAR database). Right: Tumor types caused by respective fusion oncogene. Note tumor type specificity for each fusion oncoprotein and that the FET‐NTDs replace each other as fusion partners in some entities. Only a selection of the FET family fusion oncoproteins and tumors is shown as more members are continuously discovered. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Control, Modification, Binding Assay, RNA Sequencing, Knockdown, Generated, Comparison, Selection

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: A Immunoblot analysis (IB) of histone modifications in replicate R2 of stably transfected HT1080 cell lines expressing EGFP, FUS‐DDIT3‐EGFP, or EWSR1‐FLI1‐EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. Stable replicate R1 is visualized in Fig A. B Immunoblot analysis (IB) of HT1080, and HT1080 transiently expressing FUS‐DDIT3‐EGFP or EWSR1‐FLI1‐EGFP (samples harvested after 24 or 48 h transient transfection) with antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion proteins. C, D Immunoblot analysis (IB) of HT1080 and HT1080 FUS‐DDIT3‐EGFP (C) or HT1080 EWSR1‐FLI1‐EGFP (D) (both stable expression) with antibodies against H3K27me3, H3K4me3, and histone loading control H4. Immunoblot analysis with GFP or FLI1 antibody is shown to verify expression of FET fusion protein and normal FLI1. Note that endogenous FLI1 around 50 kDa is expressed in HT1080 cell lines. E Immunoblot analysis (IB) of extracts from untreated EWS TC‐71, cells treated with 5 μM tazemetostat for 72 h and DMSO‐control. Detection using antibodies against H3K27me3, H3K4me3, and histone loading control H4 showed substantial decrease of H3K27me3 after inhibition of EZH2 with tazemetostat. F Diagrams showing amount, quantified from immunoblots in Fig A (R1) or panel (A) (R2), of H3K27me3, H3K4me3, and H3K27Ac (in relation to corresponding histone loading control H4), ratio H3K27me3/H3K4me3 as well as amount of EZH2 compared to loading control GAPDH for HT1080 EGFP, HT1080 FUS‐DDIT3‐EGFP, and HT1080 EWSR1‐FLI1‐EGFP normalized to parental HT1080. Each diagram panel shows data from one experiment. G Diagrams showing amount, quantified from immunoblots in panel (B), of H3K27me3, H3K4me3, and H3K27Ac (in relation to corresponding histone loading control H4), ratio H3K27me3/H3K4me3 as well as amount of EZH2 compared to loading control GAPDH for HT1080 FUS‐DDIT3‐EGFP and HT1080 EWSR1‐FLI1‐EGFP normalized to parental HT1080. Samples were harvested after 24‐h or 48‐h transient transfection. Each diagram panel shows data from one experiment. H, I Diagram showing amount of H3K27me3 and H3K4me3 (in relation to corresponding histone loading control H4) as well as ratio H3K27me3/H3K4me3 for HT1080 FUS‐DDIT3‐EGFP (H) quantified from panel (C) or HT1080 EWSR1‐FLI1‐EGFP (I) quantified from panel (D) (both stable expression) normalized to parental HT1080. Each diagram panel shows data from one experiment. J Diagram showing amount, quantified from immunoblots in panel (E), of H3K27me3 and H3K4me3 (in relation to corresponding histone loading control H4) as well as ratio H3K27me3/H3K4me3 for untreated EWS TC‐71, cells treated with 5 μM tazemetostat for 72 h and DMSO‐control, normalized to the untreated control. Data from one experiment. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Control, Inhibition

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of GFP‐tagged proteins co‐immunoprecipitated with BRG1. Detection with antibodies against BRG1 and DDIT3 in HT1080 cell lines with stable expression of FUS‐DDIT3‐EGFP or DDIT3‐EGFP. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for DDIT3 in HT1080 FUS‐DDIT3‐EGFP or DDIT3‐EGFP. The specific interaction (BRG1) is compared to the non‐specific interaction (negative control IgG). Mean ± SEM is shown with individual replicates indicated by circles, n = 3. Student's t ‐test, ns = not significant. Original data for all quantifications, including P ‐values, are shown as source data. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation, Expressing, Negative Control

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Verification of cell lines by RT–PCR over fusion breakpoints (brkp) of MLS 2645‐94 (FUS‐DDIT3 type II), MLS 402‐91 (FUS‐DDIT3 type I), EWS TC‐71 (EWSR1‐FLI1 type I), EWS IOR/CAR (EWSR1‐ERG), HT1080 FUS‐DDIT3‐EGFP (FUS‐DDIT3 type II), and HT1080 EWSR1‐FLI1‐EGFP (EWSR1‐FLI1 type I): list of cell line, fusion type, assay used (forward and reverse primer) and expected product size indicated.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Reverse Transcription Polymerase Chain Reaction