Journal: Journal of Biological Chemistry

Article Title: Role of Hypoxia-inducible Factor-1 in Transcriptional Activation of Ceruloplasmin by Iron Deficiency

doi: 10.1074/jbc.m000636200

Figure Lengend Snippet: FIG. 3. Binding of HIF-1 to the HRE of the Cp enhancer (by EMSA). A, Induction of complex formation by HIF-1 agonists. Hep3B cells were exposed for 8 h to 1 mM desferrioxamine (DFO), 1 mM bathophenanthroline sulfate (BPS), or 1% O2 (Hpx.). Nuclear extracts were incubated with 32P-labeled, oligonucleotide 24-mer probes con- taining either the Cp or Epo HRE. Complexes formed were resolved by 5% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the putative HIF-1, constitutive (Const.), and nonspecific (NS) complexes are indicated by arrows. B, competitor binding to show specificity of HIF-1 binding to the Cp enhancer HRE. Hep3B cells were treated with 1% O2 for 9 h, and nuclear extracts were prepared as in A. A 10-, 100-, or 1000-fold molar excess of unlabeled, annealed oligonucleotide competitor representing the wild-type (wt) Cp HRE, the mutant (mut) Cp HRE, or the Epo HRE was added to the nuclear extract reaction mixture just prior to addition of radiolabeled Cp HRE probe. The mutated sequence in the Cp HRE is underlined. C, identification of HIF-1 subunits binding to the Cp en- hancer HRE by gel supershift analysis. Hep3B cells were treated with desferrioxamine, and nuclear extracts were prepared as in A. Before subjecting extracts to electrophoresis, the mixtures containing 32P- labeled Cp HRE probe was incubated with 1 ml of anti-HIF-1a, anti- HIF-1b, or both. The supershifted complex is indicated by the open- headed arrow.

Article Snippet: For gel supershift analysis, 1 ml of rabbit monoclonal antibody against HIF-1a or rabbit polyclonal antibody against ARNT/HIF-1b (both from Novus Biologicals, Littleton, CO) was added after the initial 20-min incubation, and the solution was further incubated for 30 min at 4 °C before electrophoresis.

Techniques: Binding Assay, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Mutagenesis, Sequencing, Electrophoresis

Journal: Molecular Cancer Research

Article Title: The Opposing Effect of Hypoxia-Inducible Factor-2α on Expression of Telomerase Reverse Transcriptase

doi: 10.1158/1541-7786.mcr-07-0065

Figure Lengend Snippet: FIGURE 4. HIF-2a occupancy on the hTERT promoter, recruitment of p300, and changes in histone H3 acetylation in A498 and U251 cells under hypoxic conditions. A. Schematic presentation of the hTERT promoter and locations of the ChIP PCR amplicons relative to the translational start codon (ATG). The putative HREs in region a are indicated (16). Region b was also found to interact with HIF-1a according to Anderson et al. (10). B. The ChIP assay for the presence of HIF-2a, p300, and histone H3 acetylation at the hTERT promoter in hypoxia-treated A498 and U251 cells. The precipitated DNA was subject to PCR analysis using the primer pairs specific to the hTERT promoter regions a and b. Input is sonicated chromatin without immunoprecipitation, but reverse cross-linked and purified together with ChIP samples. HRE, hypoxia-responsive element; H and N, the cells were cultured under hypoxic and normal oxygen conditions, respectively. Ab, no antibody controls. H3-Ac, the antibody against acetylated histone H3.

Article Snippet: The membranes were probed with the specific mouse monoclonal antibody against HIF-1a or HIF-2a rabbit polyclonal antibody (Novus Biologicals), followed by a secondary anti-mouse or rabbit horseradish peroxidase–conjugated immunoglobulin G, and developed with the enhanced chemiluminescence method (Amersham).

Techniques: Sonication, Immunoprecipitation, Purification, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 1. EXTL2 mRNA expression levels after siRNA-mediated knock- down of EXTL2 in HEK293 cells. HEK293 cells were transiently transfected with three different human siRNAs (siL2A, siL2B, and siL2C) targeting EXTL2, one negative non-targeting control siRNA (siNC), and transfection reagent only (Mock) as described under “Experimental Procedures.” 24 h after trans- fection, relative mRNA levels were determined by real-time PCR and normal- ized to those of -actin. mRNA levels after siRNA treatments are expressed relative to the mock expression that was set to 1. The error bars represent the meanS.D.fromatleastfourindependenttransfections.Eachmeasurement was performed in duplicate or triplicate. The numbers above the bars show the percentage of down-regulation.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Expressing, Knockdown, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 2. Effect of siRNA-mediated knockdown of EXTL2 on HS chain length. A and B, 3H-labeled (A) or 35S-labeled (B) HS was purified from the cell surface/extracellular matrix of siRNA (siL2M, siL2A, siL2C) and the non-target- ing control siRNA-transfected (siNC) and mock-transfected (Mock) HEK293 cells 48 h after transfection as described under “Experimental Procedures.” Isolated glycosaminoglycans were digested with chondroitinase ABC and subjectedtogelchromatographyonaSuperose6column.Theretardedcom- ponents eluting at 19–23 ml correspond to material that was degraded by chondroitinase ABC. Similar results were obtained from three independent transfections. The samples in A and B were run on two different Superose 6 columns calibrated using size-defined fragments of heparin and hyaluronan. The elution positions of molecular mass standards derived from heparin (8.6 kDa) and hyaluronan (19, 30, 43 and 210 kDa) are indicated by arrows.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Knockdown, Labeling, Purification, Control, Transfection, Isolation, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 3. Subcellular localization of EXTL2 in HEK293 cells. HEK293 cells were stably transfected with full-length tGFP-EXTL2 expression plasmid. Trans- fectants were fixed and stained for EXTL2 (green) and the trans-Golgi marker TGN46 (red) as described under “Experimental Procedures.” The nuclei were counterstained with DAPI (blue).

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Staining, Marker

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 4. EXTL2 expression levels after overexpression of EXTL2 in HEK293 cells. HEK293 cells were stably transfected with the full-length tGFP- tagged EXTL2 and with vector only (Mock) as described under “Experimental Procedures.” A, mRNA levels for three clones stably overexpressing EXTL2 (L2cl.1, L2cl.2, and L2cl.3) were determined by real-time PCR and normalized to those of -actin. The mRNA levels after overexpression are expressed as -foldchangesrelativetothemockexpressionthatwassetto1.Eachmeasure- ment was performed in triplicate. The mRNA levels of EXT1, EXT2, and EXTL3 (L3) were also determined for the three EXTL2-overexpressing clones (inset). The values for EXT1, EXT2, and EXTL3 are the average values across the three overexpressing clones from two independent experiments. The error bars represent mean values S.D. B, Western blot of the same three clones (L2cl.1, L2cl.2, and L2cl.3) stably overexpressing EXTL2 as in A. Cell extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride mem- brane (“Experimental Procedures”). The blot was probed with antibodies against tGFP and -actin and visualized by chemiluminescence.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Expressing, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Real-time Polymerase Chain Reaction, Western Blot, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 6. Cell surface expression of HS on HEK293 cells overexpressing EXTL2. A–C, EXTL2-overexpressing, mock-transfected, and wild-type cells were examined for cell surface expression of HS by flow cytometry using the 10E4 antibody (A) and the 3G10 antibody before (B) and after (C) heparitinase (Hep) digestion. The histograms show the intensity of the fluorescence (per- centage of maximum (% of max)) and is representative of two independent experiments. Black line, EXTL2-overexpressing cells; dashed line, mock-trans- fected cells; dotted line, wild-type cells; filled gray profile, control (secondary antibody only). PE-A, Phycoerythrin-Area.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Expressing, Transfection, Flow Cytometry, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 7. Glycosyltransferase activities of HEK293 cells overexpressing EXTL2. Glycosyltransferase activities of cell extracts or immunopurified EXTL2, from untransfected (wt), empty vector-transfected (mock), EXTL2-transfected (L2), and EXT2-transfected HEK293 cells are shown as indicated. In A–F, oligosaccharides derived from Escherichia coli K5 capsular polysaccharide were used as acceptor substrates in transferase assays. The K5 polysac- charide has the same structure as the nonsulfated HS precursor molecule, and oligosaccharide derivatives thereof, with non-reducing terminal GlcA or GlcNAc residues served as acceptors in the GlcNAc- and GlcA-transferase reactions. C, shows the GlcNAc-transferase activity of immunopurified EXTL2 after transient transfection, whereas the other panels show enzyme activities after stable transfection. No GlcA-transferase activities were detected with immunopurified EXTL2 (data not shown). In G, oligosaccharides with a non-reducing GlcA residue, generated from defructosylated E. coli K4 capsular polysaccharide with the same structure as the nonsulfated chondroitin sulfate precursor molecule, were used as acceptor substrate in transferase assays. The error bars represent mean values S.D. from four (A) and two (B, D, and E) independent EXTL2-overexpressing clones and from one clone (F). G, representative results from one out of two independent analyses. Each clone was assayed at two different protein concentrations. *, p 0.01, **, p 0.001. ns, not significant.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Plasmid Preparation, Transfection, Derivative Assay, Activity Assay, Stable Transfection, Residue, Generated, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

doi: 10.1074/jbc.m114.631754

Figure Lengend Snippet: FIGURE 9. FAM20B mRNA expression level in HEK293 cells overexpress- ing EXTL2. FAM20B relative mRNA levels were determined by real-time PCR and normalized to those of -actin. mRNA levels are expressed as fold changesrelativetothelevelsofmock-transfected(Mock)cellsthatweresetto 1. The error bars represent mean S.D. of values from four different EXTL2 (L2)-overexpressing clones measured in triplicates. ****, p 0.0001.

Article Snippet: Alternatively, HEK293 cells were transfected using the Nucleofector electroporation kit for adherent cells (Amaxa) with a C-terminal TurboGFP (tGFP)tagged full-length human EXTL2 cDNA clone in the pCMV6AC-GFP vector (OriGene).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Clone Assay