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

Journal: Cancers

Article Title: In Vitro Diffuse Large B-Cell Lymphoma Cell Line Models as Tools to Investigate Novel Immunotherapeutic Strategies

doi: 10.3390/cancers15010235

Figure Lengend Snippet: Overview of immunotherapeutic strategies and cell lines used in evaluation of their efficacy.

Article Snippet: Tafasitamab (anti-CD19 mAb) , B-ALL cell lines: SEM, Jurkat, CEM, MOLT-16, Nalm-6 cells , [ ] .

Techniques: Bioprocessing

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: Workflow for CTC capture and PD-L1 analysis

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques:

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: Examples of PD-L1 IHC staining of FFPE sections from NSCLC patients (× 100): a PD-L1(−) negative; b positive control (× 200), c PD-L1(+) positive, < 50% immunoreactive, d PD-L1(+) positive, > 50% immunoreactive

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques: Immunohistochemistry, Positive Control

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: PD-L1 expression levels in ten cancer cell lines and in PD-L1 positive and negative control cells from the BioINK staining kit

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques: Expressing, Negative Control, Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: Example images of PD-L1 positive and negative staining patterns

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques: Negative Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: CTC detection rate and PD-L1% in treatment-naïve NSCLC patients

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques:

Journal: Cancer Immunology, Immunotherapy

Article Title: Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer

doi: 10.1007/s00262-019-02344-6

Figure Lengend Snippet: 2 × 2 Confusion matrix and performance measures

Article Snippet: The PD-L1 positive control cell line and the PD-L1 negative control cell line were obtained from InCellDx, Inc. (IncellDx, Menlo Park, CA, USA).

Techniques: