Journal:

Article Title: A Ubiquitous ?-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

doi: 10.1091/mbc.E04-01-0060

Figure Lengend Snippet: HAβ5-tubulin overexpression disrupts microtubules. CHO (A), NIH 3T3 (B), and HeLa (C) cells were transfected with pTOP/HAβ5 plasmid DNA and grown 3 days in αMEM. The cells were then processed for immunofluorescence using antibodies specific for the HA-tag (green fluorescence) and α-tubulin (red fluorescence). The specimens were also stained for DNA (blue fluorescence). Nontransfected cells are red (α-tubulin staining only), but cells that express HAβ5-tubulin are yellow (low-to-moderate HAβ5-tubulin content) or green (high HAβ5-tubulin content). Note that high levels of HAβ5 expression frequently cause extensive fragmentation of microtubules (arrows). Bar (A), 20 μm.

Article Snippet: Transfection and Isolation of Stable Transfected Cell Lines CHO tTA 6.6a cells expressing the tetracycline regulated transactivator ( Gonzalez-Garay et al., 1999 ) were seeded into a 35-mm tissue culture dish containing a sterile coverslip and transfected with pTOP/HAβ5 using Lipofectamine reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer.

Techniques: Over Expression, Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining, Expressing

Journal:

Article Title: A Ubiquitous ?-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

doi: 10.1091/mbc.E04-01-0060

Figure Lengend Snippet: Western blot analysis of HAβ5-tubulin expression in stably transfected cell lines. Clonal CHO cell lines with stable expression of HAβ1-tubulin (lanes 1 and 2), HAβ5-tubulin (clone HAβ5c8, lanes 3 and 4; clone HAβ5c12, lanes 5 and 6), and mutant HAβ1(L215H)-tubulin (lanes 7 and 8) were selected in G418 and analyzed for tubulin production using a mAb that recognizes the amino terminal region of both transfected (HAβ) and endogenous (β) β-tubulin. An antibody recognizing actin (A) was also included to act as a control for loading. The cells were maintained in 1 μg/ml tetracycline and then grown 24 h in the presence (+) or absence (-) of the antibiotic before analysis. Note that there is little or no expression of the HA-tagged proteins in presence of tetracycline.

Article Snippet: Transfection and Isolation of Stable Transfected Cell Lines CHO tTA 6.6a cells expressing the tetracycline regulated transactivator ( Gonzalez-Garay et al., 1999 ) were seeded into a 35-mm tissue culture dish containing a sterile coverslip and transfected with pTOP/HAβ5 using Lipofectamine reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Mutagenesis

Journal:

Article Title: A Ubiquitous ?-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

doi: 10.1091/mbc.E04-01-0060

Figure Lengend Snippet: Microtubule organization in stably transfected cells. Cell lines HAβ1 (A), HAβ5c12 (B), HAβ5c8 (C), and HAβ1(L215H) (D) were grown 3 days in the absence of tetracycline to induce expression of the transfected cDNA. The cells were extracted in a microtubule stabilizing buffer and stained with an antibody specific for transfected HA-tagged β-tubulin (green fluorescence) and with a dye for nuclear DNA. Although the DNA stained blue in immunofluorescence as in Figure 1, here we pseudocolored it red to improve the contrast of the figure. Note the sparse microtubule network and fragmented microtubules (arrows) in the cell line (C) with high HAβ5-tubulin expression. Insets in A and C show mitotic cells from the same cultures. Bar (A), 20 μm.

Article Snippet: Transfection and Isolation of Stable Transfected Cell Lines CHO tTA 6.6a cells expressing the tetracycline regulated transactivator ( Gonzalez-Garay et al., 1999 ) were seeded into a 35-mm tissue culture dish containing a sterile coverslip and transfected with pTOP/HAβ5 using Lipofectamine reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer.

Techniques: Stable Transfection, Transfection, Expressing, Staining, Fluorescence, Immunofluorescence

Journal:

Article Title: A Ubiquitous ?-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

doi: 10.1091/mbc.E04-01-0060

Figure Lengend Snippet: HAβ5-tubulin incorporates efficiently into microtubules. Cell lines HAβ1 (lanes 1 and 2), HAβ5c8 (lanes 3 and 4), HAβ5c12 (lanes 5 and 6), and HAβ1(L215H) (lanes 7 and 8) were grown 48 h in αMEM, lysed in a microtubule stabilizing buffer, and centrifuged. Pellet fractions (lanes 1, 3, 5, and 7) containing polymerized tubulin, and supernatant fractions (lanes 2, 4, 6, and 8) containing soluble tubulin were run on SDS-polyacrylamide gels, electroblotted onto nitrocellulose membranes, and probed with an antibody recognizing the amino terminus of both endogenous (β) and transfected (HAβ) β-tubulins. The ratio of HAβ/β is shown for each of the fractions. Note that the amount of polymerized tubulin is very low for HAβ5c8 (lane 3), yet the ratio of exogenous to endogenous tubulin reflects the abundance of the transfected tubulin in the cells.

Article Snippet: Transfection and Isolation of Stable Transfected Cell Lines CHO tTA 6.6a cells expressing the tetracycline regulated transactivator ( Gonzalez-Garay et al., 1999 ) were seeded into a 35-mm tissue culture dish containing a sterile coverslip and transfected with pTOP/HAβ5 using Lipofectamine reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer.

Techniques: Transfection

Journal:

Article Title: A Ubiquitous ?-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

doi: 10.1091/mbc.E04-01-0060

Figure Lengend Snippet: Expression of HAβ5-tubulin in paclitaxel selected cells. CHO tTApur 6.6a cells were transfected with pTOP/HAβ5 and selected in G418 in presence of tetracycline to obtain stably transfected cells. A portion of the total G418-resistant population (lane 1) was then reselected in αMEM without tetracycline but containing 100 ng/ml (117 nM) paclitaxel for 10 days (lane 2). Cells surviving the paclitaxel selection were compared with the G418-resistant cells by growing both cell populations 24 h in αMEM, separating the proteins on SDS gels, and staining Western blots with an antibody that recognizes the amino terminal region of both transfected (HAβ5) and endogenous (β) β-tubulin. The ratio of transfected to endogenous β-tubulin (HAβ5/β) for both cell populations is shown.

Article Snippet: Transfection and Isolation of Stable Transfected Cell Lines CHO tTA 6.6a cells expressing the tetracycline regulated transactivator ( Gonzalez-Garay et al., 1999 ) were seeded into a 35-mm tissue culture dish containing a sterile coverslip and transfected with pTOP/HAβ5 using Lipofectamine reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer.

Techniques: Expressing, Transfection, Stable Transfection, Selection, Staining, Western Blot

Journal: Scientific Reports

Article Title: Novel CMKLR1 Inhibitors for Application in Demyelinating Disease

doi: 10.1038/s41598-019-43428-8

Figure Lengend Snippet: Comparing CMKLR1 antagonist α-NETA and FDA-approved Tecfidera (dimethyl fumarate, DMF) in suppressing experimental autoimmune encephalomyelitis (EAE). ( A ) Structure and features of α-NETA and DMF. ( B ) EAE was induced in C57BL/6 mice by active immunization with MOG 35–55 /CFA. Mice received α-NETA (10 mg/kg daily; n = 8 mice), DMF (10 mg/kg daily, n = 4 mice)), or vehicle control (10% captisol, n = 4 mice) beginning at the time of disease induction and were monitored daily for clinical disease as follows: 0, normal; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb and hind limb paralysis; 5, dead. Day of onset of clinical signs (assumes worst case scenario d29 onset for 2 mice in the α-NETA group). Mean + SEM, *p < 0.05, **p < 0.01 by one-way ANOVA. ns, not significant. ( C ) Mean clinical score - SEM. *p < 0.0001 by F-test (extra sum of squares) comparing the shapes of the 4 th order polynomial curves for each treatment group, rejecting the null hypothesis that one curve fits all data sets. ( D ) The integrated clinical score over time for each animal was calculated as area under the curve (AUC). Mean + SEM, *p < 0.05, **p < 0.01 by one-way ANOVA.

Article Snippet: DiscoverX PathHunter® CHO-K1 CMKLR1 β-Arrestin Cells were grown in RPMI lacking phenol red (Gibco, Life Technologies), supplemented with L-glutamine, penicillin-streptomycin, sodium pyruvate, non-essential amino acid, BCS, hygromycin, and G418-sulfate.

Techniques: Control

Journal: Scientific Reports

Article Title: Novel CMKLR1 Inhibitors for Application in Demyelinating Disease

doi: 10.1038/s41598-019-43428-8

Figure Lengend Snippet: SAR of α-NETA analogs for CMKLR1 inhibition. Thin arrows indicate structural features leading to increased potency.

Article Snippet: DiscoverX PathHunter® CHO-K1 CMKLR1 β-Arrestin Cells were grown in RPMI lacking phenol red (Gibco, Life Technologies), supplemented with L-glutamine, penicillin-streptomycin, sodium pyruvate, non-essential amino acid, BCS, hygromycin, and G418-sulfate.

Techniques: Inhibition

Journal: Scientific Reports

Article Title: Novel CMKLR1 Inhibitors for Application in Demyelinating Disease

doi: 10.1038/s41598-019-43428-8

Figure Lengend Snippet: SAR of tertiary amines on CMKLR1 inhibition.

Article Snippet: DiscoverX PathHunter® CHO-K1 CMKLR1 β-Arrestin Cells were grown in RPMI lacking phenol red (Gibco, Life Technologies), supplemented with L-glutamine, penicillin-streptomycin, sodium pyruvate, non-essential amino acid, BCS, hygromycin, and G418-sulfate.

Techniques: Inhibition

Journal: Scientific Reports

Article Title: Novel CMKLR1 Inhibitors for Application in Demyelinating Disease

doi: 10.1038/s41598-019-43428-8

Figure Lengend Snippet: SAR of aryl group on CMKLR1 inhibition.

Article Snippet: DiscoverX PathHunter® CHO-K1 CMKLR1 β-Arrestin Cells were grown in RPMI lacking phenol red (Gibco, Life Technologies), supplemented with L-glutamine, penicillin-streptomycin, sodium pyruvate, non-essential amino acid, BCS, hygromycin, and G418-sulfate.

Techniques: Inhibition, Chemotaxis Assay

Journal: Glycobiology

Article Title: Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand

doi: 10.1093/glycob/cwaa034

Figure Lengend Snippet: Native PSG1 and recombinant PSG1 made in CHO-K1 cells bind to Gal-1 with similar affinity in a carbohydrate-dependent manner. (A) Native PSG1 was coated on wells and after blocking, 2 μg/mL of Fc-tagged mutated Gal-1 (dmGal-1), which cannot bind to LacNAc structures due to two amino acid substitutions, or wild type Gal-1 were added. After washing, binding was detected with HRP-conjugated anti-Fc Ab. (B) rGal1 was coated on wells and after blocking, native PSG1 (4 μg/mL) was added in the presence of 100 mM lactose or 100 mM sucrose, as indicated. PSG1 binding was detected with an anti-PSG1 biotin-labeled MAb. (C) SPR sensogram of the interaction of Gal-1 (black line) and hIgG-Fc used as negative control (grey line) at 1 μM with immobilized native PSG1. The beginning of the Gal-1 and hIgG-Fc injections are indicated with an arrow head, and the association and dissociation phases are specified. A control surface without immobilized protein was used as a control for potential unspecific binding to the biosensor surface and subtracted from the binding of Gal-1 to the PSG1 surface. (D) Sensorgrams of the interaction of Gal-1 with native PSG1. Serial dilutions of Gal-1 ranging from 1 to 0.01 μM were injected during 3 min over a CM5 biosensor chip with immobilized native PSG1. SPR sensorgrams for each protein concentration are shown as grey lines while the fitted data are shown as black lines. (E) Sensorgrams of the interaction of Gal-1 with recombinant PSG1 represented as in (B). (F) Sensorgrams of the interaction of Gal-1 at 0.25 μM with immobilized recombinant PSG1 in the absence (black) or presence of 1.43 mM of sucrose (grey) or increasing concentrations of lactose (from light to dark red).

Article Snippet: PSG1-His, PSG1-Fc and the PSG1N-Fc were harvested from the supernatant of stably transfected CHO-K1 cell lines grown in a C2003 hollow fiber cell culture cartridge (FiberCell Systems, Frederick, MD).

Techniques: Recombinant, Blocking Assay, Binding Assay, Labeling, Negative Control, Injection, Protein Concentration

Journal: Glycobiology

Article Title: Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand

doi: 10.1093/glycob/cwaa034

Figure Lengend Snippet: Differences in binding of two glycoforms of PSG1 to Gal-1 and ability to mediate protection from oxidative inactivation. (A) Recombinant PSG1 produced in CHO cells (lane 1) or N-acetylglucosaminyltransferase I (GnTI)-deficient cells (lane 2) were purified using a HisTrap column followed by gel filtration chromatography. Purified proteins were separated on SDS-PAGE and stained with GelCode Blue. (B) Recombinant PSG1 generated in CHO-K1 cells or GnTI-deficient cells was coated on wells and after blocking, 2 μg/mL of Fc control protein or Fc-tagged recombinant human Gal1 (hGal1-Fc) were added. Binding was detected with HRP-conjugated anti-Fc Ab. Fc control values were subtracted from hGal-1Fc values. (C) SPR sensorgram showing the interaction of Gal-1 at 1 μM with PSG1 produced in CHOK1 or GnTI-deficient cells. (D) BSA, PSG1 made in CHO-K1 cells or PSG1 made in GnTI-deficient cells were coupled to CnBr-Sepharose beads. Gal-1 was applied to each resin, followed by the addition of 20 mM H2O2. The oxidation reaction was stopped with catalase and Gal-1 was eluted from each resin with 100 mM Lactose, dialyzed against PBS and its concentration determined by ELISA. ASF or BSA were coated on wells and after blocking, oxidized Gal-1 recovered from each resin was added to the ASF or BSA-coated wells at 0.25 μg/mL. Gal-1 binding was detected with anti-Gal-1 biotin-labeled Ab. Results are expressed as percentage of Gal-1 inactivation after H2O2 treatment and values are derived from absorbance values of each sample treatment by the formula listed in the materials and method section.

Article Snippet: PSG1-His, PSG1-Fc and the PSG1N-Fc were harvested from the supernatant of stably transfected CHO-K1 cell lines grown in a C2003 hollow fiber cell culture cartridge (FiberCell Systems, Frederick, MD).

Techniques: Binding Assay, Recombinant, Produced, Purification, Filtration, Chromatography, SDS Page, Staining, Generated, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Derivative Assay

Journal: Glycobiology

Article Title: Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand

doi: 10.1093/glycob/cwaa034

Figure Lengend Snippet: Ability of PSG1 to compete the binding of Gal-1 to cell surface receptors and to soluble fibronectin. (A) FN and 3% BSA were coated on wells and after blocking, Gal-1 pre-incubated with PBS (−), PSG1 generated in CHO-K1 cells or in GnTI-deficient cells at the indicated concentrations was added to the wells. Binding of Gal-1 was detected with a biotin-labeled anti-Gal-1 Ab followed by streptavidin-HRP. The numbers shown in the figure represent the O.D. values obtained following subtraction of the O.D. values of the BSA-coated wells. (B) Jurkat cells were incubated with biotin-labeled Gal-1 in the presence or absence of PSG1 generated in CHO-K1 cells or in GnTI-deficient cells at the indicated concentrations. Binding of biotinylated Gal-1 to the cells was detected with streptavidin-APC. (C) Swan 71 cells were incubated in FACS buffer or in FACS buffer containing 2 or 20 μg/mL PSG1 as indicated. After washing, Gal-1 bound to the cells was detected with biotin-labeled anti-Gal-1 Ab followed by streptavidin-APC. Cells incubated with biotin-labeled isotype control Ab followed by streptavidin-APC were used as control.

Article Snippet: PSG1-His, PSG1-Fc and the PSG1N-Fc were harvested from the supernatant of stably transfected CHO-K1 cell lines grown in a C2003 hollow fiber cell culture cartridge (FiberCell Systems, Frederick, MD).

Techniques: Binding Assay, Blocking Assay, Incubation, Generated, Labeling