Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Amplification

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Control, Immunohistochemistry, Expressing, Staining

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 1 EPHA2 protein structure. EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand- binding domain and TK domain by us recently

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 3 EPHA2 gene amplification in SSC and MPM samples. Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 2 EPHA2 gene sequencing chromatograms. Representative sequencing chromatograms of the mutation region in normal (N) and tumor (T) samples. Boxes indicate the heterozygous mutation in the tumor sample. a Novel mutation (R159G and R876C) and known single-nucleotide polymorphism (SNPs) (P350T, G391R, M631T, R876H, and R890Q) were detected in SSC tumor tissue. b Novel mutation (T140I, D184Y, T647M, and A859D) and known SNPs (R195C, D232G, T647T, and R876H) were detected in MPM tumor tissue. R159G, T140I, D184Y, R195C, and D232G were found in exon 3, P350T was found in exon 5, T647T/M were found in exon 10, M631T was found in exon 11, A859D/A and R876H/R were found in exon 15., and R876C, R876H, and R890Q were found in exon 16

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Sequencing, Mutagenesis

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 4 EPHA2 expression in MPM cell lines and in MPM and SCC tumor tissues. a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 6 Cell cytotoxicity of SCC EPHA2 isogenic cells with chemo drugs and small molecular inhibition. BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Inhibition, Mutagenesis, Plasmid Preparation

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 5 Cell viability and migration of HEK293 EPHA2 isogenic cells. a Cell viability was measured every 24 h over 72 h. b Cell migration was measured by wound healing assay at 0, 2, and 5 h

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Migration, Wound Healing Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 7 Cell cytotoxicity of HEK293 EPHA2 isogenic cells. a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 8 Molecular dynamics simulations. a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å3) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Fig. 9 The EPHA2 mechanistic model. The model depicts how in the A859D mutant loss of Y772 phosphorylation leads to enhanced tumorigenesis in comparison to T647M mutant

Article Snippet: EPHA2 knockdown EPHA2 knockdown was performed by transfecting cells with EPHA2 shRNA (OriGene, Rockville, MD) as per manufacturer’s instructions.

Techniques: Mutagenesis, Phospho-proteomics, Comparison

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: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: EPHA2 is expressed on the cell surface of undifferentiated mouse ESCs and downregulated during differentiation. (A) Relative gene expression of Epha2 mRNA in mouse ESCs (D3) cultured without LIF for 0, 3, 5, and 7 days. (B) Immunoblotting of EPHA2 protein of mouse ESCs cultured as in (A). (C) EPHA2 protein levels in (B) normalized to α-Tubulin. (D) Immunofluorescent staining of mouse ESCs. The cells were cultured with or without LIF for 7 days. Bars; 200 μm. (E, F) Flow cytometric analysis of EPHA2 protein on the cell surface of mouse ESCs. Living cells were stained with an EPHA2 antibody. The propidium iodide + dead cells were removed from the analysis. FSC and SSC profiles (E) and the histogram of EPHA2-AF488 levels (F) of the gated cells in (E) were shown. Statistical analyses in (A) and (C) were calculated by Dunnett’s test. Data were graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01; *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Gene Expression, Cell Culture, Western Blot, Staining

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Knock-down of Epha2 induces spontaneous differentiation of mouse ESCs. (A) qRT-PCR analysis of Epha2 mRNA after knock-down in mouse ESCs (D3). The cells were infected with Epha2 shRNA retrovirus and cultured with or without LIF for 7 days. (B) Images of phase contrast and alkaline phosphatase (AP) staining of Epha2 KD mouse ESCs cultured in ES maintenance medium with LIF for 5 days. Bars; 200 μm. (C) qRT-PCR analysis of undifferentiated state-specific marker genes in Epha2 KD mouse ESCs. (D) Immunofluorescent staining of Epha2 KD mouse ESCs cultured with LIF for 7 days. Bars; 100 μm. (E) Culture conditions of Epha2 KD mouse ESCs after infection with Epha2 shRNA retrovirus. One day after infection with Epha2 shRNA virus, mouse ESCs were cultured with or without 2i in the presence of G418 and LIF for 4 days. (F) Phase contrast images of Epha2 KD mouse ESCs cultured as in (E). Bar; 200 μm. (G) qRT-PCR analysis of Epha2 and undifferentiated state-specific marker genes in Epha2 KD mouse ESCs cultured with 2i. All qRT-PCR analyses were performed with 3 independent biological replicates and graphed as means ± SE. The significant differences were calculated by Tukey test in (A and C) and t -test in (G). * P < .05; ** P < .01, *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Knockdown, Quantitative RT-PCR, Infection, shRNA, Cell Culture, Staining, Marker, Virus

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Expression of EPHA2 in heterogenous subpopulation of human PSCs. (A) qRT-PCR analysis of EPHA2 and undifferentiated state-specific marker genes during EB-based random differentiation of human iPSCs (201B7) without basic FGF. Statistical analysis was done by Dunnett’s test comparing to day 0 and graphed as means ± SE of 3 independent experiments. * P < .05; ** P < .01. (B) Representative flow cytometric plots of living human iPSCs stained with EPHA2 antibody-conjugated with AFF488. Gate3 and Gate4 were sorted as EPHA2 − and EPHA2 + cell populations, respectively. See also . (C) Feature plots of EPHA2 and undifferentiated state-specific genes in publicly available undifferentiated human ESC H1 and H9 data subsets from GSE75748 scRNA-seq dataset. Note that EPHA2 expression in hESCs was heterogeneous. Normalized expression levels were plotted. (D, E) Immunofluorescent staining of human iPSC cultured on SyntheMax II-coated plate with StemFit medium. The cells were fixed with paraformaldehyde in PBS and permeabilized. Bars; 200 μm. (F) qRT-PCR analysis of fractioned EPHA2 + and EPHA2 - subpopulations. EPHA2 + cells express higher OCT4 and NANOG , than EPHA2 − cells. Means ± SE of 3 independent experiments were shown. Statistical significance was defined as * P < .05 by t -test.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Quantitative RT-PCR, Marker, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Transplantation of EPHA2 + cells into immune-deficient mice formed tumors in vivo. (A) Immunofluorescent staining of mouse EBs differentiated into hepatocyte lineages. Expression of an early hepatocyte marker AFP at day 10 and a mature marker ALB at day 14 were detected. Bars; 200 μm. (B) Depletion of undifferentiated ES colonies after removal of EPHA2 + cells from EBs. Oct4-egfp ESCs were differentiated by EB formation for 10 and 14 days. EPHA2 + cells were removed from EBs using anti-EPHA2 antibody-bound MACS after dissociation with trypsin/EDTA. The residual cells were cultured in ES maintenance medium with LIF for 7 days. The alkaline phosphatase (AP) activity was visualized by incubating with AP substrate. Bar; 2 cm. (C) The number of EGFP + cell colonies in (B). Statistical analysis was done by Tukey test and graphed as means ± SE of 4 independent experiments. (D) Scheme of in vivo transplantation experiment. Mouse ESCs (D3) were differentiated into hepatocyte linages and the EBs were dissociated by EDTA treatment. The cells were transplanted into SCID mice after depletion of EPHA2 + cells by MACS. (E) Decreased teratoma formation after transplantation of EPHA2 − cells. White arrowheads indicate teratomas. (F) H&E staining of teratomas formed in the testicular subcutaneous tissue without MACS procedure. Typical cell types of 3 germ layers were confirmed. Bar; 500 μm. (G) Typical teratoma formation after transplantation of dissociated EB at day 10 through hepatic portal vein. White arrowheads indicate teratomas. (H) Quantification of teratoma formation in (E) and (G). Statistical analysis was done by Chi-square test, * P < .05, ** P < .01.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Transplantation Assay, In Vivo, Staining, Expressing, Marker, Cell Culture, Activity Assay

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Co-expression of EPHA2 with OCT4 in EBs during human iPSC differentiation into hepatocyte. (A, B) Relative gene expression of undifferentiation and differentiation markers during hepatic induction. Statistical analysis was done by Dunnett’s test against day 0 and graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01, *** P < .001. (C) Immunofluorescent staining of EBs at days 5, 8, and 10. Bars; 200 μm. (D) Enlarged images of EB at day 5 in (C). Bars; 50 μm. (E, F) Quantification of immune-positive cells in . Box plot showing the percentage of EPHA2 + cells among SOX17 + or OCT4 + cells (E). Box plot showing the percentage of EPHA2 + and TRA1-81 + cells among OCT4 + cells (F). Each box represents 1st quartile, median, and 3rd quartile, and whiskers show the minimum and maximum values. Ten images of independent EBs were analyzed. Total count of DAPI + nuclei per image were between 1 × 10 3 and 2 × 10 3 . Statistical significance was defined by Tukey test of SOX17 and OCT4, respectively in (E) and t -test between EPHA2 and TRA1-81 in (F). **P p < .01, *** P < .001, N.S; no significance between 3 with P > .05.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Gene Expression, Staining