Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: In vitro characterization of IgG25 and IgG28. (a) FACS-based whole cell binding assay was performed with IgG25 and IgG28 on human MiaPaCa2 and (b) on mouse MC38-CEA cells to determine the apparent Kd. Mean Fluorescence Intensities (MFIs) obtained with IgG25 and IgG28 over the logarithm of their molar concentration (LogM) are reported. (c) Binding competition experiments of IgG25 and IgG28 with ephrinA1 on MiaPaCa2 cells, a control isotypic IgG1 antibody (Ctrl IgG) was used as negative control. IgG25 (filled circles), IgG28 (empty circles), Ctrl IgG (filled triangles). (d) EphA2 immunoprecipitation from lysates of cells treated with Ctrl IgG, IgG25, IgG28 and ephA1-Fc, followed by Western Blot with antiphosphotyrosine antibody. After stripping, the same filter was probed with EphA2 antibody as loading control. (e) FACS-based whole cell binding with IgG25 and IgG28 on mouse N2A cells transiently transfected with expression vectors coding for members of Eph A receptor family (EphA1, EphA2, EphA3, EphA4, EphA5, and EphA7).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vitro, Cell Binding Assay, Fluorescence, Concentration Assay, Binding Assay, Control, Negative Control, Immunoprecipitation, Western Blot, Stripping Membranes, Transfection, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25- and IgG28-mediated EphA2 internalization and degradation in MiaPaCa2 cells. (a) EphA2 internalization in response to IgG25, IgG28, and ephrinA1-Fc treatment. mAbs and ephrinA1-Fc labeled in red while cell nuclei in blue. Images were acquired at 20× magnification. Localization was revealed 1 hour after incubation on cells either at 4°C or at 37°C. (b) Time course Western blot analysis of EphA2 degradation after treatment with control IgG, IgG25, IgG28, and ephrinA1-Fc; anti- β -actin was used as loading control. (c) Densitometric analysis of the level of EphA2 expression measured by Western Blot in cells treated with control IgG (asterisks), IgG25 (squares), IgG28 (triangles), and ephrinA1-Fc (circles). Data are expressed as percentage of EphA2 expression over time.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Labeling, Incubation, Western Blot, Control, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 downstream signaling in MiaPaCa2 cells treated with IgG25 and IgG28. Cell lysates of MiaPaCa2 were assayed by Western Blotting with antiphospho-Akt, antiphospho-ERK, and anti-phospho FAK (Tyr 576). Antibodies directed to total Akt, total FAK, and actin were used as loading controls.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Western Blot

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: Effect of EphA2 antibodies on angiogenesis. (a) Representative CD31 immunostaining of paraffin embedded tumor section (magnification bar = 20 m). (b) Quantitative analysis of CD31 staining in tumors treated with control IgG, IgG25, and IgG28. The average data obtained from the analyses of two tumors selected from each group are reported as the percentage of CD31 + area in each entire section. In all panels, asterisks indicate statistically significant differences with respect to the control group (Student's t -test; P <.05).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Immunostaining, Staining, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 protein expression in tumors from treated mice. (a) Western Blot analysis with anti-EphA2 antibodies of lysates from five tumors treated with control IgG, with IgG25, or six tumors treated with IgG28. (b) Densitometric analysis of the ratio between EphA2 levels revealed by anti-EphA2 antibody in nonsaturating conditions and actin expression measured with antiactin antibody. Data on y axis are expressed as arbitrary units.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25 and IgG28 modulate in vivo EphA2 downstream signaling: Western Blot analyses of tumor lysates with antiphospho FAK (Tyr 576) and anti-phospho Akt. Antibodies to total Akt, total FAK, and actin were used as loading controls. P values were calculated with respect to average densitometric value of group treated with control IgG.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vivo, Western Blot, Control

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Expressing, Western Blot

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Phospho-proteomics, Immunoprecipitation, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Control, Inhibition, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Activity Assay, Expressing, Western Blot, Cytotoxicity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Activation Assay

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test