Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Ectopic LIF expression (left panel) or treatment with rhLIF (right) enhanced lactate production of different breast cancer cell lines analyzed by measuring lactate levels in the medium using a lactate assay kit. B LIF neutralization antibody (LIF neu-ab) blocked the promoting effect of LIF on lactate production in MCF7, MDA-MB 231 and T47D cells. C Knockdown of endogenous LIF decreased lactate production in MDA-MB 231 cells. D , E Ectopic LIF expression enhanced glycolysis rates ( D ), whereas knockdown of endogenous LIF decreased glycolysis rates ( E ) in different breast cancer cell lines as calculated by ECAR measured by using a Seahorse analyzer. F The diagram of glycolysis. G – I The fold change of inter-metabolites in glycolysis resulted from ectopic LIF expression in MCF7 and MDA-MB 231 cells ( G ), rhLIF treatment in MCF7 cells ( H ) or LIF knockdown in MDA-MB 231 cells ( I ) as determined by LC/MS metabolomics analysis. J The fold change of inter-metabolites in glycolysis induced by ectopic LIF expression in MCF7 xenograft tumors determined by LC/MS metabolomics analysis. Data are presented as mean ± SD ( n = 3/group). * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t -test.

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Expressing, Lactate Assay, Neutralization, Knockdown, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Glut1 mRNA levels were detected by quantitative real-time PCR (qPCR) in MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression. B , C Ectopic LIF expression ( B ) or rhLIF treatment (100 ng/ml for 12 h) ( C ) promoted endogenous Glut1 PM translocation in MCF7, MDA-MB 231, and T47D cells as determined by Western-blot assays. D Knockdown of LIF decreased endogenous Glut1 PM translocation in MDA-MB 231 cells. E Ectopic LIF expression increased the PM translocation of ectopically expressed Myc-Glut1 in MCF7, MDA-MB 231 and T47D cells as determined by Western-blot assays. F LIF neutralization antibody (LIF neu-ab) largely abolished exogenous Glut1 PM translocation promoted by LIF. G The rhLIF treatment promoted exogenous Glut1 PM translocation in cells. H Knockdown of LIF decreased Myc-Glut1 PM translocation in MDA-MB 231 cells. I Ectopic LIF expression promoted Myc-Glut1 PM translocation (left panels) while knockdown of LIF decreased Myc-Glut1 PM translocation (right panels) in MDA-MB 231 cells as determined by IF staining assays. Scale bar, 10 μm. J Ectopic LIF expression promoted the PM translocation of Myc-Glut1 in MCF7, MDA-MB 231, and T47D cells as determined by flow cytometry assays. Left panels: representative images of flow cytometry analysis. Right panels: quantifications of relative fluorescence intensity of Myc-Glut1 on the cell membrane normalized with total Myc-Glut1 fluorescence intensity in cells. In A , J data are presented as mean ± SD. n = 3/group. * p < 0.05; ** p < 0.01; NS: non-significant; unpaired Student’s t -test. Uncropped Wes t ern-blot images are shown in Supplementary Fig .

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Translocation Assay, Western Blot, Knockdown, Neutralization, Staining, Flow Cytometry, Fluorescence, Membrane

Journal: JCI Insight

Article Title: EGFR -mutant transformed small cell lung cancer harbors intratumoral heterogeneity targetable with MEK inhibitor combination therapy

doi: 10.1172/jci.insight.197008

Figure Lengend Snippet: ( A ) Heatmap of cell viability after 5 days’ treatment with 1 μmol/L of each single agent (rows) per various lung cancer cell lines (columns) relative to DMSO control. Commercially available agents used are in . ( B ) Bar graph of IC 50 values of various indicated lung cancer cell lines in response to trametinib. IC 50 of NE and non-NE tSCLC cell lines is shown in red and blue, respectively. ( C ) Dose-response curve of trametinib in tSCLC cell lines (blue: NE, red: non-NE). Cell viability was assessed by CTG after 120 hours ( n = 6, technical replicate, mean ± SD). ( D ) NE tSCLC cell viability was assayed by CTG following treatment with indicated concentration of LIF and IL-6 for 5 days. ( E ) qPCR analysis of LIF mRNA expression levels in non-NE tSCLC cell lines treated with 10 nmol/L trametinib 2 and 6 hours. Gene expression was normalized to GUSB and shown as relative to that of DMSO-treated control ( n = 3, technical replicate, mean ± SD). ****= P ≤ 0.001 by 2-way ANOVA with Šídák’s multiple comparisons test. ( F ) Various cell lines were analyzed for LIF expression by ELISA. Data are presented as mean ± SD ( n = 3. Technical replicates). ( G ) GFP-expressing DFCI112F and DFCI190F cells were incubated with CM from their corresponding adherent cell line pairs pretreated with either LIF neutralizing antibody (500 ng/mL) or control IgG and monitored in real time via IncuCyte imager for green fluorescence intensity. ( H and I ) GFP expressing DFCI112F ( H ) and DFCI190 ( I ) cells were incubated with CM from their adherent cell line pairs pretreated with siRNA against LIF or unrelated sequence and monitored via IncuCyte imager. LIF knockdown efficiency was tested using ELISA ( n = 3, technical replicate, mean ± SD). ( J ) The schema illustrating the interaction between NE and non-NE tSCLC cells through LIF.

Article Snippet: LIF neutralizing antibody (AF-250-NA), IL-6 neutralizing antibody (AF-206-NA), and control antibody (AB-108-C) were purchased from R&D Systems.

Techniques: Control, Concentration Assay, Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence, Sequencing, Knockdown

Journal: PLoS ONE

Article Title: Leukemia Inhibitory Factor Enhances Endometrial Stromal Cell Decidualization in Humans and Mice

doi: 10.1371/journal.pone.0025288

Figure Lengend Snippet: A–D. LIF immunolocalized strongly to the decidua (d) and also to the luminal epithelium (le). E–H. LIFR immunolocalized strongly to the decidua. Bottom row, negative controls to sections above. The same negative is shown for both antibodies as the primary antibodies shown were both raised in goat. n = 3; m. myometrium; s, stroma.

Article Snippet: LIF and LIFR were immunolocalised as described above except that the non-immune block used was: LIF: 10% normal goat plus 2% normal mouse serum and LIFR: 10% normal horse and 2% mouse serum; and two LIF antibodies were used to confirm the specificity of LIF staining (rabbit anti-human LIF antibody as above and in and goat anti-human LIF antibody, R&D systems; 10 μg/ml).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: Association of Cul2 with BAF250b. (A) BAF250b associates specifically with Cul2 and not Cul5. T7 immunoprecipitates of transiently transfected T7-BAF250b with Myc-Cul2 (lanes 1, 2, 5, and 6) or Myc-Cul5 (lanes 3, 4, 7, and 8) were washed in a buffer containing 0.3 M KCl and analyzed by immunoblotting with an antibody to T7 (α-T7) or Myc. (B) Bacterially expressed Cul2 interacts with BAF250bCTD. GST pulldown assays were performed as for Fig. ​Fig.1C1C with His-Cul2 expressed in E. coli. (C) Cul2 association with the BAF250b C terminus is BC box dependent. Lysates from cells transiently transfected with T7-BAF250bNTD, T7-BAF250bCTD, or T7-BAF250bCTDBC* and with Myc-Cul2 were immunoprecipitated with T7 antibody, washed in a buffer containing 0.3 M KCl, and analyzed by immunoblotting with an antibody to T7 or Myc. (D) Cul2 association with BAF250b N terminus is ARID dependent. Lysates from cells transiently transfected with T7-BAF250bNTD or the T7-BAF250b N terminus with the ARID deletion (ΔA) and Myc-Cul2 were immunoprecipitated with T7 antibody, washed in a buffer containing 0.3 M KCl, and analyzed by immunoblotting with an antibody to T7 or Myc. (E) Endogenous Cul2 association with BAF250b is BC box dependent. T7 immunoprecipitates of transiently transfected T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA and BC*) were washed in a buffer containing 0.3 M KCl and analyzed by immunoblotting with an antibody to T7 or endogenous Cul2. (F) A schematic summarizing the interactions between BAF250b constructs and Elo C and Cul2. n.d., not determined. (G) Association of endogenous BAF250a/b and Cul2. BAF250a and BAF250b were immunoprecipitated from HEK293 cells and probed for Cul2 and BAF250. The bands corresponding to BAF250a and BAF250b are indicated by ▪ and ▪▪, respectively. (H) Endogenous Roc1 is enriched in immunopurified Flag-BAF250b complex. Flag-BAF250b immunoprecipitated from induced (+) or uninduced (−) 16e cell nuclear extracts was washed in a buffer containing 0.3 M (lanes 3 and 4) or 0.5 M (lanes 5 and 6) KCl and eluted with a Flag peptide. Eluted material was analyzed by immunoblotting for endogenous Roc1 or Flag-BAF250b.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Construct

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BAF250 contains a BC box and associates with Elo C. (A) Schematic diagram of BAF250b (GenBank accession no. AF521671). 16e, amino acids 419 to 2165; NTD, 419 to 1547; CTD, 1518 to 2165. Putative BC boxes identified by MAST scanning of human BAF250a/b and Drosophila Osa are shown. Amino acid numbering in the second column corresponds to the GenBank accession numbers listed in parenthesis. Sequences of consensus and putative BC boxes are listed in the third column, with key residues highlighted in bold. (B) The BAF250a/b C-terminal domain associates with Elo C. Upper two panels, cells were transiently transfected with Flag-Elo C alone (lanes 1, 3, and 5) or together with T7-BAF250aCTD (amino acids 1635 to 2285, GenBank accession no. O14497) (lanes 2, 4, and 6). Lysates were immunoprecipitated (IP) with α-T7 antibody, washed in a buffer containing 0.3 M KCl (lanes 3 and 4) or 0.5 M KCl (lanes 5 and 6), and analyzed by immunoblotting with an antibody to T7 or Flag. Lower two panels, the same experiment was performed with T7-BAF250bCTD. (C) Bacterially expressed Elo C interacts with BAF250b CTD. Polyhistidine (His)-tagged Elo C was expressed in E. coli, and the lysate was mixed with purified GST-BAF250bCTD (HR2) (lane 3) or control GST-TAFH (lane 2). Immunoblotting (WB) with an antibody to the poly-His tag shows specific binding of Elo C to BAF250bCTD. HR2 represents the second conserved region in BAF250bCTD, which contains the BC box (residues 1786 to 2165, boxed in gray in Fig. ​Fig.1A).1A). (D) BAF250bCTD interaction with Elo C is BC box dependent. Flag antibody immunoprecipitates of transiently transfected Flag-Elo C with T7-BAF250bNTD, T7-BAF250bCTD, or T7-BAF250bCTD with the BC box mutation (BC*) were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag. *, nonspecific band. (E) BAF250b interacts with Elo C in a BC box-dependent manner. T7 antibody immunoprecipitates of transiently transfected vector, T7-BAF250b, or T7-BAF250bBC* with Flag-Elo C were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Purification, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BC* degradation by the proteasome is Cul2 dependent. (A) Stability of BAF250b and BC* protein. HeLa cells transiently transfected with T7-BAF250bWT (lanes 1 to 3) or T7-BAF250bBC* (lanes 4 to 6) were treated with vehicle (−) or cycloheximide (CHX) for the times indicated. Whole-cell extracts were immunoblotted for T7-250b and endogenous BRG1. (B) BC* degradation is exacerbated by overexpression of Cul2. HeLa cells were transiently transfected with T7-BAF250b (lanes 1 to 6) or T7-BAF250bBC* (lanes 7 to 12) and Myc-Cul2 (lanes 1 to 12). Cells were treated with MG132 or CHX for the times indicated. Whole-cell extracts were immunoblotted for T7, Myc, or endogenous BRG1. (C) The degradation of BAF250bBC* is exacerbated by Cul2 but not Cul5. HeLa cells were transiently transfected with T7-BAF250bBC* (lanes 1 to 12) and Myc-Cul5 (lanes 1 to 6) or Myc-Cul2 (lanes 7 to 12). Cells were treated with MG132 or CHX for the times indicated. Whole-cell extracts were immunoblotted for T7, Myc, or endogenous Elo A (loading control).

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Over Expression

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: BAF250b is ubiquitinated and degraded by the proteasome. (A) BAF250bCTD BC* mutants are degraded during immunoprecipitation. T7 antibody immunoprecipitates of transiently transfected T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA and BC*), with Myc-Cul2, were washed in a buffer containing 0.5 M KCl and 0.1% NP-40 and analyzed by immunoblotting with an antibody to T7 or Myc. The film exposure for the input panels was shorter than that for the IP panels. (B) MG132 and NEM stabilize BAF250b mutant proteins in the presence of Myc-Cul2. HeLa cells were transiently transfected with T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA, BC*) and with Myc-Cul2, treated with 5 μM MG132, and harvested in the presence of NEM. BAF250b proteins were immunoprecipitated with an antibody to T7 and probed for Myc-Cul2. (C) BAF250bCTDBC* is autoubiquitinated. T7 antibody immunoprecipitates of cells transiently transfected with vector (−) (lanes 1, 5, and 9), T7-BAF250bCTDWT (+) (lanes 2, 6, and 10), T7-BAF250bCTDBC* (lanes 3, 7, and 11), or T7-BAF250bCTDBC** (lanes 4, 8, and 12) and Myc-Cul2 (lanes 1 to 12) were washed in a buffer containing 0.3 M KCl and 0.5% NP-40 and immunoblotted for ubiquitin (endogenous), Myc, and T7.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: The BAF250b complex contains an E3 ligase activity that ubiquitinates histone H2B at K120. (A) Immunopurification of Flag-BAF250b complex. A silver-stained gel of SWI/SNF purified from a HeLa cell line, 16e, induced to express Flag-BAF250b is shown (lane 2). Purification from uninduced (−) 16e cells is shown as a negative control (lane 1). The SWI/SNF subunits identified by mass spectrometry are indicated. (B) Identification of SWI/SNF subunits by immunoblotting. Endogenous BRG1, BAF170, BAF155, and E3 ligase subunit Cul2 were detected in Flag-BAF250b-immunopurified material eluted with Flag peptide from induced cells (lane 4). An antibody to Flag was used to detect Flag-BAF250b. (C) The BAF250b complex monoubiquitinates histone(s) in a ubiquitination assay. An in vitro nucleosome ubiquitination assay mixture was reconstituted with the Flag-BAF250b complex (lanes 2 to 4), control Flag-eluted material (lane 1), or recombinant Ring1b (lane 5) and HA-ubiquitin (lanes 1 to 5). The products were immunoblotted (WB) with an antibody to HA and H2B. Ten microliters of eluted material correlates roughly with each lane in the silver-stained gel shown in panel A. Oligonucleosomes were omitted from the reaction in lane 4. Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2. (D) Bacterially expressed BAF250bCTD interacts with histone H2B. GST-BAF250bCTD (HR2) (lane 2) and control GST-TAFH (lane 3) proteins were expressed and purified from E. coli and incubated with histones extracted from HeLa cells. Immunoblotting (WB) with an antibody to H2B shows specific binding of H2B to BAF250bCTD. The lower panel shows the upper portion of the same gel stained with Coomassie blue. (E) The BAF250b complex monoubiquitinates histone H2B on K120. An in vitro nucleosome ubiquitination assay was performed as for panel C with the Flag-BAF250b complex (lanes 2 and 4), control material (lane 1), or recombinant Ring1b (lanes 3 and 5). The products were immunoblotted with an antibody to HA and H2B. The nucleosomes used were reconstituted in vitro with wild-type H2B (lanes 1 to 3) or H2B with lysine-120 mutated to alanine (H2BK120A) (lanes 4 and 5). Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Activity Assay, Immu-Puri, Staining, Purification, Negative Control, Mass Spectrometry, Western Blot, Ubiquitin Assay, In Vitro, Recombinant, Incubation, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B ▿

doi: 10.1128/MCB.00540-09

Figure Lengend Snippet: Global levels of H2B-Ub and H3K4me3 are decreased following knockdown of BAF250a and BAF250b by RNAi. (A) Knockdown of BAF250 by RNAi. Whole-cell extracts from HEK293 cells transiently transfected with siRNAs against luciferase (control), BAF250a, BAF250b, or both BAF250a and BAF250b (a+b) were immunoblotted with an antibody to BAF250b. *, nonspecific band used as a loading control. (B) The same extracts as for panel A were immunoblotted with an antibody to BAF250a. (C) Decreased global H2B-Ub levels upon BAF250 siRNA knockdown. Acid extracts from HEK293 cells depleted of BAF250a (lane 3), BAF250b (lane 2), or BAF250a+b (lane 4) were immunoblotted with an antibody to H2B. The band migrating above 20 kDa in the upper panel corresponds to the size of H2B-Ub. The upper and lower panels are from the same gel. The blot was separated for probing because of the difference in signal intensity between H2B-Ub and H2B. (D) The same extracts as for panel C were immunoblotted with an antibody specific to H3K4me3 (upper panel) and H3 (lower panel). (E) BAF250a is a positive regulator of the HoxA9 gene. Quantitative RT-PCR of HoxA9 mRNA levels in HEK293 cells transiently transfected with siRNAs targeting luciferase (control), BAF250a, BAF250b, or both BAF250a and BAF250b (a+b) is shown. Error bars indicate standard deviations.

Article Snippet: Thirty microliters of Flag-BAF250b complexes (purified from induced 16e cells [16e+]) or the negative-control eluant (purified from uninduced 16e cells [16e−]) was dialyzed into ubiquitination buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 2 mM NaF, 0.6 mM dithiothreitol [DTT]) at 4°C for 1 h. After adjusting for changes in volume during dialysis, equivalent amounts of 16e−, Flag-BAF250b complexes, or recombinant Flag-tagged Ring1b were incubated with 2 mM ATP, 0.1 μg E1 (Boston Biochem), 0.5 μg E2 (UbcH5c; Boston Biochem), 1 μg hemagglutinin antigen-tagged ubiquitin (HA-Ub) (Boston Biochem), and 5 to 10 μg of in vitro -reconstituted oligonucleosomes (wild type or H2BK120A) in 30- to 40-μl reaction volumes for 30 min at 37°C.

Techniques: Transfection, Luciferase, Quantitative RT-PCR

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: Active gp130/STAT3 pathway in PANC-1 sphere cells. ( A ) Real-time qPCR analysis of IL-6 , LIF , IL-6R , LIFR , and gp130 in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. The results presented are normalized to values obtained for adherent cells (value = 1). Results are presented as means ± SD from three independent experiments. ( B ) FACS analysis of IL-6R, LIFR, and gp130 expression in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Controls are indicated by thin lines with gray color. ( C ) Cell surface levels of IL-6R, LIFR, and gp130 expression in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Mean fluorescence intensities (MFIs) relative to those of adherent cells are presented. Results are presented as means ± SD from three independent experiments. ( D ) Western blot analysis of p-gp130, gp130, p-STAT3, and STAT3 was performed in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Relative band intensity is provided. ** p < 0.01.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Cell Culture, Expressing, Fluorescence, Western Blot

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: The correlation of gp130/STAT3 pathway-related factor expression with overall survival and H19 expression correlated with gp130/STAT3 pathway-related factors in patients with PDAC. Each survival curve was presented according to the online database GEPIA. ( A ) IL-6 , ( B ) LIF , ( C ) IL-6R , ( D ) LIFR , ( E ) gp130 (IL-6ST) , ( F ) JAK1 , ( G ) STAT3 , ( H ) TGF β-RII (TGFBR2) , ( I ) MT1-MMP (MMP14) , and ( J ) H19 . ( K ) The correlation of H19 and gp130/STAT3 pathway-related factor expression in patients with PDAC was assessed using Spearman rank correlation analysis according to the online database GEPIA.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Expressing

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: Schematic representation of autocrine/paracrine IL-6 or the LIF/gp130/STAT3 pathway in PDAC sphere cells. In PDAC sphere cells in which CSCs are enriched (CSC-like cells), binding of autocrine/paracrine IL-6 or LIF to each receptor (IL-6R or LIFR, respectively) induces gp130 homodimerization or LIFR/gp130 complex formation to thereby activating JAKs followed by phosphorylation of gp130, ultimately leading to STAT3 activation. This pathway contributes to the maintenance of stemness features and expression of MT1-MMP and TGFβ-RII. Expression of TGFβ-RII via the gp130/STAT3 pathway affects TGF-β1/Smad signaling to promote EMT induction. Additionally, p-STAT3 can access the active promoter region of H19 and contribute to its transcription. Therefore, autocrine/paracrine IL-6 or the LIF/gp130/STAT3 pathway in PDAC CSC-like cells is believed to eventually lead to invasion and metastasis, both of which are hallmarks of malignancy.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Binding Assay, Phospho-proteomics, Activation Assay, Expressing