Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P<0.001 by one-way ANOVA. (C–D)PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.5 μg/ml ephrin-A 5Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. (C) The histogram shows the average area of the cells normalized to the value obtained for the Fc-treated cells. Error bars represent standard errors from three wells. The average cell areas in cells treated with ephrin-A1 Fc and compound were compared to that in cells treated only with ephrin-A1 Fc by one-way ANOVA and Bonferroni’s post test, showing that 76D10 significantly (***P<0.001) inhibits ephrin-A1-dependent cell retraction at concentrations between 100 and 25 μM. The effect of ephrin-A1 was reverted completely by 100 μM 76D10 and partially by 50 and 25 μM (comparison between Fc and ephrin-A1 Fc treated samples at each compound concentration yielded P values of >0.05 for 100μM, <0.05 for 50 μM and <0.001 for 25μM 76D10. (D) Representative images of cells stained with rhodamine-phalloidin to label actin filaments (red) and DAPI to label nuclei (blue). Scale bar = 50 μm.

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Activation Assay, Control, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Comparison, Concentration Assay, Staining

Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits ephrin- and TNFα-induced tyrosine phosphorylation and capillary-like tube formation in HUVE cells. (A) Cells plated on Matrigel were treated with the indicated concentrations of 76D10 or DMSO and imaged 18 hours later. The number of polygons present in each picture and the average tube length were quantified. The histograms show averages from 4 independent experiments and the error bars represent the standard errors. **P<0.01 and ***P<0.001 by one-way ANOVA and Dunnett’s post test. (B) HUVE cells were left unstimulated or stimulated with 20 nM TNFα for 2 hours in the presence of the indicated concentrations of 76D10. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphA2. (C) MTT assay to determine the number of viable HUVE cells after growth in the presence of the indicated concentrations of 76D10 for 1 or 3 days. Only DMSO was used in the “0 μM” sample, as a control. The histogram shows average absorbance at 570 nm in the presence of 76D10 normalized to the absorbance in the absence of the compound. Error bars represent standard error from 3 measurements in each of two experiments. *P<0.05 by one-way ANOVA and Dunnett’s post test for the comparison to cells not treated with compound (0 μM).

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Phospho-proteomics, MTT Assay, Control, Comparison

Journal: Molecular Therapy

Article Title: Combinational Targeting Offsets Antigen Escape and Enhances Effector Functions of Adoptively Transferred T Cells in Glioblastoma

doi: 10.1038/mt.2013.185

Figure Lengend Snippet: Heterogeneous antigen expression in glioblastoma (GBM) . Flow cytometric analysis of single cell suspensions of primary GBM excision samples and U373-GBM cell line costained for HER2, IL-13Rα2, and EphA2 on > 100,000 gated events ( Supplementary Figure S1 describes the gating strategy) shown in ( a ) representative histograms and ( b ) dot plots.

Article Snippet: Surface staining of tumor cells was done using mouse anti-HER2.APC (BD Biosciences), mouse anti-IL-13Rα2.PE (Abcam, Cambridge, MA), and goat anti-EphA2 with rabbit anti-goat IgG.FITC (Southern Biotech, Birmingham, AL) antibodies.

Techniques: Expressing

Journal: Molecular Therapy

Article Title: Combinational Targeting Offsets Antigen Escape and Enhances Effector Functions of Adoptively Transferred T Cells in Glioblastoma

doi: 10.1038/mt.2013.185

Figure Lengend Snippet: Hierarchy of expression of various antigenic permutations in primary glioblastoma (GBM) . Multiaxial graph depicting prevalence of target antigens in various tumor cell sub-populations studied using coimmunofluorescence for HER2, IL-13Rα2, and EphA2 of six serially diagnosed GBMs surgical specimens. Significantly, more cells fell in populations expressing any two antigen permutations (versus single antigen only) by comparing means using a one-tailed unequal variance t -test. Expression of any of the three antigens was not significantly more inclusive of the tumor bulk in the patient cohort studied. Representative IFC captures are shown in Supplementary Figures S2 and S3 . Data are analyzed in a mathematical model in .

Article Snippet: Surface staining of tumor cells was done using mouse anti-HER2.APC (BD Biosciences), mouse anti-IL-13Rα2.PE (Abcam, Cambridge, MA), and goat anti-EphA2 with rabbit anti-goat IgG.FITC (Southern Biotech, Birmingham, AL) antibodies.

Techniques: Expressing, One-tailed Test