Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, MCA205 tumor cells were treated with various doses of 17-DMAG for 24h in vitro, then lysed, with EphA2 and control β-actin protein expression subsequently monitored by western blotting as described in Materials and Methods. NC; negative control lysate from EphA2neg B16 melanoma cells. In B, proteasome inhibitor (MG-132), but not lysosome inhibitor chloroquine (CLQ), blocks 17-DMAG (500 nM)-induced degradation of EphA2 protein in MCA205 tumor cells. In C, treatment of MCA205 cells with 17-DMAG at the indicated doses for 24h (or 48h, data not shown) did not affect MHC class I expression on tumor cells. In D, 17-DMAG-treated EphA2+ MCA205 cells were better recognized versus control, untreated tumor cells by anti-EphA2 CD8+ T cells (developed from EphA2 −/− mice, per Fig. S1 and Materials and Methods) in CD107 translocation assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: In Vitro, Control, Expressing, Western Blot, Negative Control, Translocation Assay

Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, C57BL/6 mice bearing established MCA205 tumors (day 18; ~100 mm2 mean tumor size) were left untreated or they were administered 17-DMAG (10, 15 or 25 mg/kg/day for up to 10 days via oral gavage) and tumor size (mean +/minus; SD, 5 animals/group) monitored longitudinally. *p < 0.05; **p < 0.01 (ANOVA) for 15 or 25 mg/kg/day versus 10 mg/kg/day or untreated; not significant (ANOVA) for 15 versus 25 mg/kg/day. In B, tumors were excised on the indicated day after initiating treatment, and single cell suspensions of enzymatic tumor digests analyzed for immune cell infiltrates by flow cytometry as described in Materials and Methods. Tumor cells isolated from enzymatic digests (per Fig. 2B) were also analyzed as target cells for anti-EphA2 CD8+ T effector cells generated from EphA2 −/− mice (see Fig. S1) as monitored using CD107 translocation (C) and IFN-γ secretion (D) assays as described in Materials and Methods. All data are representative of those obtained in 3 independent experiments. For panels B–D, *p < 0.05; **p < 0.01 (ANOVA) versus all other determinations.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Flow Cytometry, Isolation, Generated, Translocation Assay

Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, MCA205 tumor-bearing mice (5 mice/group) were left untreated or they were treated for 5 days with orally-administered 17-DMAG (15 mg/kg/day), with tumor growth then monitored over a 4 week period. B, EphA2 protein expression in tumors harvested from 17-DMAG-treated versus untreated mice was analyzed longitudinally by western blotting as outlined in Materials and Methods. C, Tumor cells from untreated or 17-DMAG-treated mice were analyzed at the indicated time points for their ability to be recognized by anti-EphA2 CD8+ T cells generated from EphA2 −/− mice (see Fig. S1) in CD107 translocation and IFN-γ secretion assays, as described in Materials and Methods. D, Single cell suspensions from harvested tumor digests were analyzed by flow cytometry for the indicated T cell, DC and MDSC phenotypes. All data are representative of those obtained in 3 independent experiments. *p < 0.05 (t-test) for treated versus untreated controls.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Expressing, Western Blot, Generated, Translocation Assay, Flow Cytometry

Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, C57BL/6 mice bearing established EphA2+ MCA205 sarcomas (s.c. right flank) remained untreated, or they were treated with DC-based vaccines (s.c., left flank on days 0 and 7 of the treatment regimen) that contained or lacked EphA2 peptide epitopes, alone or in combination with 17-DMAG (15 mg/kg/day on the first 5 days of the treatment regimen by oral gavage). Tumor size (mean +/minus; SD) is reported in mm2. All the mice in the DC/EphA2 + 17-DMAG-treated group rendered tumor-free (80%) were rechallenged (s.c., right flank) with MCA205 tumor cells on day 30 of the experiment (as indicated by arrow with “R” inset) and monitored through day 60 after treatment initiation. B, CD8+ TIL recovered from tumors on day 14 after treatment initiation were assessed for their ability to recognize syngenic control DC pulsed with no peptide or DC pulsed with the EphA2671-679 + EphA2682-689 peptides. After 48h incubation, cell-free supernatants were analyzed for IFN-γ content by ELISA. Response to DC (no peptide) was < 50 pg/ml) in all instances. C, Single-cell suspensions of enzymatically-digested day 14 (post-treatment initiation) tumors were analyzed by flow cytometry for the indicated T cell, DC and MDSC phenotypes as described in Materials and Methods. Each filled circle represents data from an individual animal in a given control or treatment cohort, with the mean of data indicated by a gray bar for each cohort. All data are representative of those obtained in 3 independent experiments. *p < 0.05; **p < 0.01 (ANOVA) versus all other cohorts.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Vaccines, Control, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, C57BL/6 mice bearing established s.c. B16 melanomas (right flank) were left untreated or treated as outlined in Fig. 5A, with tumor size (mean +/minus; SD) reported in mm2 followed for up to 30 days. *p < 0.001 (ANOVA) versus all other cohorts. B, Day 14 (post-treatment initiation) tumors were harvested and tissue sections analyzed by immunofluorescence microscopy and Metamorph quantitation for co-expression of CD31 (i.e. VEC) and EphA2 proteins as described in Materials and Methods. *p < 0.05 (ANOVA) versus all other cohorts. Anti-EphA2 CD8+ T cells isolated from the spleens of immune EphA2 −/− mice (panel C; as outlined in Fig. S1), or TIL from B16 tumor-bearing animals treated with combined DC/EphA2 peptide vaccination + 17-DMAG (panel D; per Fig. 5A) were analyzed for reactivity against flow-sorted CD31+ VEC isolated from the tumors of B16-bearing animals left untreated or treated for 6 days with DC/EphA2 vaccine only, 17-DMAG or DC/EphA2 vaccine + 17-DMAG. CD31+ kidney VEC were also flow sorted from animals treated for 6 days with DC/EphA2 vaccine + 17-DMAG to discern “autoimmunity” of T cells against tumor-uninvolved VEC. In C, the MHC class I-restricted nature of VEC recognition by CD8+ T cells was assessed by inclusion of anti-class I or isotype control mAb per culture well, as described in Materials and Methods. For panels C and D, *p < 0.05 (t-test) versus control antibody treatment or untreated controls, respectively. All data are representative of those obtained in 3 independent experiments.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Immunofluorescence, Microscopy, Quantitation Assay, Expressing, Isolation, Control

Journal: Cancer Research

Article Title: Combination therapy with HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment to improve recruitment of therapeutic T cells 1

doi: 10.1158/0008-5472.CAN-12-0538

Figure Lengend Snippet: A, C57BL/6 mice bearing established s.c. MCA205 sarcomas (right flank) were left untreated or they were treated with 17-DMAG (15 mg/kg/day provided orally on the first 5 days of the treatment regimen) +/minus;adoptive transfer (i.v. tail vein on day 4 of the treatment regimen) of 5 × 106 CD8+ T cells isolated from EphA2 −/− mice previously vaccinated with syngenic DC (control T cells) or DC loaded with the EphA2671-679 + EphA2682-689 peptides. Tumor size was monitored longitudinally and is reported (mean +/minus; SD) in mm2 from 5 mice/group. All animals treated with combined anti-EphA2 (immune) T cell + DMAG therapy required euthanasia due to core necrosis on day 14 after treatment initiation. In B, day 14 untreated or treated tumors underwent enzymatic digestion, with single-cells analyzed by flow cytometry for the indicated T cell, DC and MDSC phenotypes as described in Materials and Methods. Each filled circle represents data from an individual animal/cohort with the data mean indicated by the gray bar. In C, CD8+ TIL harvested from day 14 (post-treatment initiation) tumors were analyzed for IFN-γ secretion in response to EphA2 peptide-pulsed syngenic (control) DC by ELISA as outlined in Materials and Methods. *p < 0.05; **p < 0.01 (ANOVA) versus all other cohorts. All data are representative of those obtained in 3 independent experiments.

Article Snippet: The EphA2 + MCA205 sarcoma and EphA2 neg B16 melanoma (H-2 b ) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Adoptive Transfer Assay, Isolation, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: EphA2 expression and correlation with survival. A and B, immunohistochemical analysis of EphA2 staining in patient tumor tissue sections. C, Kaplan-Maier analysis of EphA2 expression versus survival. Left panel: patients stratified by EphA2 expression levels, where low and high indicate expression relative to mean expression. Right panel, patients stratified by lung cancer histology subtypes. D, immunoblot analysis of EphA2 expression in various cell lines. Lysates from various cell lines were obtained, run on SDS-PAGE, and immunoblotted with either anti-EphA2 (1) or anti-actin (2) antibody. The cell lines are: A549, SK-LU-1, H1703, H358, H1993, H661, SW1573, H522, H226, H1437, H1838, H1975, and H2170.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: Tumor histology and frequency of G391R mutation A summary of the frequency of the EphA2 G391R mutation in lung cancer tumor tissues and cell lines, demonstrating that this mutation occurred exclusively in squamous cell carcinomas.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: EphA2 WT and EphA2 G391R mutation promote anchorage-independent growth. A, immunoblot analysis of EphA2 expression in control, EphA2 wild-type (WT), and EphA2 G391R mutant transfectants. Human EphA2 cDNA (Origene) or human EphA2 cDNA containing the G391R mutation cloned into the pCDNA6.2 vector (Invitrogen), which contains antibiotic resistance genes for selection of stable transfectants in mammalian cells, were transfected into BEAS-2B cells using FuGENE HD (Roche Life Sciences), and cultured in the presence of blasticidin for more than 2 weeks to select only plasmid-containing cells. Cell lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-EphA2 (1) or anti-actin (2) antibody. B and C, analysis of anchorage-independent growth in control, empty vector (EV), EphA2 wild-type (WT), and EphA2 G391R mutant transfectants. There is a statistically significant increase in soft agar colony formation with WT and G391R transfectants with the G391R having the highest colony forming capacity. A549 cells were used as a positive control.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis, Western Blot, Expressing, Control, Clone Assay, Plasmid Preparation, Selection, Transfection, Cell Culture, SDS Page, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: EphA2 WT and EphA2 G391R mutation regulate focal adhesion dynamics. A, immunocytochemical analysis of focal adhesions and the actin cytoskeleton using anti-vinculin antibody (red) and phalloidin (green) in control, empty vector (EV), EphA2 wild-type (WT), and EphA2 G391R SNP transfectants. Immunofluorescence of BEAS-2B cells, with representative untransfected cells depicted in the top row, empty vector cells in the second from the top row, WT-EphA2 cells in the second from bottom row, and G391R-EphA2 in the bottom row. Phenotypically, the EphA2-overexpressing cells tended to be more elongated and contain invadopodia, suggesting a more malignant cellular behavior. In addition, cell size tended to be increased in these EphA2-overexpressing cells. The focal adhesions tended also to have a larger area, which was quantitated and shown to be increased in EphA2-overexpressing cells (see B). B, graphical representation of mean area per focal adhesion (y axis) for control BEAS-2B, empty vector (EV), EphA2 wild-type (WT), and EphA2 G391R SNP transfectants; n = 9 per condition.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis, Control, Plasmid Preparation, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: EphA2 and EphA2 G391R mutation promote invasion through activation of Src, cortactin, and p130Cas. A, graphical representation of the percent invasion (y axis) of either control, EphA2 WT, or EphA2 G391R mutant BEAS-2B cells as determined by using the QCM ECMatrix Cell Invasion Assay (Millipore), which measures cell invasion through a 8-μm pore filter covered with basement membrane components. Cells were added to the upper chamber in 0.2% medium, and 10% medium was added to the lower chamber. The cells were analyzed for invasion through basement membrane into the bottom chamber after 24 h. Statistical significance is indicated by bars above each experimental condition; n = 5 per condition. B and C, immunoblotting of BEAS-2B cell lines (control, empty vector (EV), EphA2 wild-type (WT), EphA2 G391R mutation, with and without Ephrin-A1 (1 μg/ml)) and its effect on downstream signaling. B, cell lysates from Ephrin-A1-treatment (0, 5, and 15 min) were obtained, run on SDS-PAGE, and immunoblotted with anti-EphA2, anti-phospho-EphA2 (pTyr594) or anti-actin antibody. C, cell lysates from Ephrin-A1-treatment (0 and 15 min) were obtained, run on SDS-PAGE, and immunoblotted with anti-phospho-Src (pY418) (1), anti-phospho-cortactin (pY421) (2), anti-phospho-p130Cas (pY165) (3), pY249 (4), pY410 (5), anti-EphA2 (6), or anti-actin (7) antibody. D, graphical representation of the ratio of phospho-protein immunoreactivity versus total EphA2 immunoreactivity from experiments described in C. Standardized average gray values for immunoreactive bands of total EphA2, pY418Src, pY421Cortactin, pY165p130Cas, pY249p130Cas, and pY410p130Cas were obtained from BEAS-2B control, empty vector (EV), EphA2 wild-type (WT), and EphA2 G391R mutant cells treated with Ephrin-A1 for 0 or 15 min, and ratios were calculated. The asterisks indicate a statistically significant difference (p < 0.05) between untreated and Ephrin-A1-treated cells; n = 3 per condition.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis, Activation Assay, Control, Invasion Assay, Membrane, Western Blot, Plasmid Preparation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: p130Cas is critical for invasiveness in cells harboring EphA2 G391R. A, immunoblot analysis of BEAS-2B cell lysates indicating complete knockdown of Src, cortactin, and p130Cas protein expression with siRNA treatment. BEAS-2B cells were transfected with siRNA (Santa Cruz Biotechnology) using siPORTamineTM as the transfection reagent (Ambion). Cells (∼40% confluent) were serum-starved for 1 h followed by incubation with 250 nm of target siRNA (or scramble siRNA or no siRNA) for 6 h in serum-free media. The serum-containing media was then added (10% serum final concentration) for 42 h. Cell lysates were obtained, run on SDS-PAGE, and immunoblotted with anti-Src (1), anti-cortactin (2), anti-p130Cas (3), or anti-actin antibody. B, graphical representation of invasion assay results with WT-EphA2 and G391R-EphA2 cells lines showing significant difference in inhibition of invasion between the groups in cells transfected with siRNAs against p130Cas. Knockdown of Src and cortactin significantly reduced cellular invasion by ∼80% with similar effects in WT and G391R cells. However, with p130Cas silencing, the G391R had significantly less (p < 0.05) invasive function compared with the WT-EphA2-overexpressing cells, siRNA#1 refers to siRNA purchased from Santa Cruz Biotechnology, siRNA#2 refers to siRNA purchased from Thermo Scientific; n = 5 per condition.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Knockdown, Expressing, Transfection, Incubation, Concentration Assay, SDS Page, Invasion Assay, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: The EphA2 G391R mutation increases cell survival through activation of the mTOR pathway. A, immunoblotting of BEAS-2B cell lines (controls, WT-EphA2, SNP (G391R)-EphA2), with and without ephrin-A1 (1 μg/ml) for 15 min and its effect on mTOR signaling. Cell lysates were obtained, run on SDS-PAGE and immunoblotted with anti-phospho-mTOR (pSer2448) (1) or (pSer2481) (2), anti-mTOR (3), anti-phospho-p70 S6 kinase (pThr389) (4), anti-p70 S6 kinase (5), anti-vinculin (6), anti-EphA2 (7), or anti-actin (8) antibody. B, graphical representation of the ratio of phospho-protein immunoreactivity versus total EphA2 immunoreactivity from experiments described in A. Standardized average gray values for immunoreactive bands of total EphA2, pSer2448mTOR, pSer2481mTOR, and pThr389 p70 S6K were obtained from BEAS-2B control, empty vector (EV), EphA2 wild-type (WT), and EphA2 G391R mutant cells treated with Ephrin-A1 for 0 or 15 min, and ratios were calculated. The asterisks indicate a statistically significant difference (p < 0.05) between untreated and Ephrin-A1-treated cells; n = 3 per condition. C, graphical representation of the percent survival (y axis) of control, empty vector (EV), EphA2 WT, or EphA2 G391R mutant BEAS-2B cells in serum-free media with or without 500 ng/ml Fas ligand treatment for 24 h. EphA2-overexpressing cells displayed significantly increased survival rates (p < 0.05). Furthermore, the mutant (G391R) EphA2 had even higher survival rate when compared with the WT-EphA2 cells (p < 0.05). Untransfected, EV, and EphA2 WT-transfected BEAS-2B cells showed decreased cell survival due to increased Fas ligand-mediated apoptotic events. However, EphA2 G391R-transfected cells showed minimal cell death despite the pro-apoptotic signal.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis, Activation Assay, Western Blot, SDS Page, Control, Plasmid Preparation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation *

doi: 10.1074/jbc.M109.075085

Figure Lengend Snippet: G391R enhances sensitivity to mTOR inhibition. Cell survival of BEAS-2B control, empty vector (EV), EphA2 wild-type (WT), or EphA2 G391R mutant-expressing cells in culture was assayed in the presence of varying rapamycin concentrations. Survival drops sharply with a 10−5 m (10 μm) rapamycin dose; this effect is more pronounced in cells harboring the G391R mutation. Statistical significance between WT and G391R cells is indicated for 10−6 m (1 μm) and 10−5 (10 μm) rapamycin concentrations.

Article Snippet: The following antibodies were used for immunoblotting: anti-EphA2, anti-phospho-cortactin (Tyr 421 ), and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-EphA2 antibody (Tyr 594 , Cell Applications, Inc.); anti-p130 Cas and anti-phospho-p130 Cas (either tyrosines 165, 249, or 410), anti-phospho-Src (Tyr 418 ), anti-mTOR, anti-phospho-mTOR (serines 2448 or 2481), anti-p70 S6 kinase, anti-phospho-p70 S6 kinase (Thr 389 ), and anti-vinculin antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Control, Plasmid Preparation, Mutagenesis, Expressing

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 is overexpressed in osteosarcoma cells. ( A ) Canine osteosarcoma cells Abrams, Eva, McKinley, Gracie, D17, Payton, and Igler, as well as non-malignant canine osteoblast cells (CnOb), were lysed and EphA2 expression was analyzed by Western blotting using an anti-EphA2 antibody. Tubulin expression was used as a loading control. EphA2 expression was quantified by densitometry, normalized on corresponding tubulin controls, and presented as percentages relative to osteoblast control (100%). The graph summarizes three independent experiments. ( B ) EphA2 expression in human osteosarcoma cells U2OS, SAOS2, MG-63, SJSA1, and 143B, as well as in non-malignant human osteoblast cells (hOb), was analyzed as in A. The graph summarizes three independent experiments. ( C ) EphA2 expression in the indicated canine osteosarcoma cell lines, a commercially available canine osteoblast cell line (CnOb), and 3 in-house preparations of canine primary osteoblasts (cOb-1-3) isolated from femoral bone samples were assessed by Western blotting and quantified as in A. The graph presents EphA2 signal intensity as arbitrary units (AU). * p < 0.05, ** p < 0.01; n.s. statistically not significant.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Expressing, Western Blot, Control, Isolation

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 promotes osteosarcoma cell proliferation. ( A ) Indicates that canine and human osteosarcoma cells were transduced with a specific shRNA targeting EphA2 (shA2) or with a non-silencing, scrambled shRNA (NS, control). EphA2 expression was then assessed by Western blotting using a specific anti-EphA2 antibody. ( B ) Abrams-NS and Abrams-shA2 cells were grown on glass coverslips to 70% confluency, fixed with 4% paraformaldehyde, and stained with anti-EphA2 (green), rhodamine phalloidin (cytoskeleton, red) and DAPI (cell nucleus, blue). Staining with a matching non-specific IgG was shown as specificity control. Fluorescent images were taken using an Olympus IX83 microscope at 200× magnification. Scale bar, 100 µm. ( C ) Indicates that cells were seeded in 96-well plates (2 × 10 3 cells per well, n ≥ 6) and cultured for 48 h. To assess cell viability, resazurin was added to the wells, and the fluorescence was measured after 4 h using a Varioskan LUX plate reader. Data are shown as mean ± SD. Experiments were repeated three times. *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Transduction, shRNA, Control, Expressing, Western Blot, Staining, Microscopy, Cell Culture, Fluorescence

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects osteosarcoma cell motility and invasion. ( A ) Human 143B-NS and 143B-shA2, and canine Eva-NS and Eva-shA2 cells were seeded into 6-well plates (5 × 10 5 cells per well) in triplicates and cultured to form confluent monolayers. Using a 200 µL pipette tip, a wound/scratch was made in each monolayer, and wells were rinsed with medium to remove cell debris. Wound closure was monitored over time by imaging the same area of each wound (2 per scratch) using an inverted microscope at 40× magnification. Wound width in each image was measured using Microsoft PowerPoint. Graphs represent percentages of wounds closed at each timepoint relative to wound width at 0 h. Scale bar, 100 µm. ( B ) The indicated canine ( top ) and human ( bottom ) cells were seeded into transwell inserts (2 × 10 4 cells per insert) in serum-free media ( n = 3 per group). Cell culture media with 10% FBS was added to the lower chamber of each insert, and cells were incubated at 37 °C for 24 h. For assessing cell migration after 24 h, cells residing on the upper side of each insert porous membrane were removed by a cotton swab, and migrated cells at the lower surface of the membrane were fixed with methanol, stained with crystal violet, and counted. For cell counting, five selected fields of each insert were imaged at 100× magnification, and the average number of cells per field was calculated. Graphs represent percentages of migrated EphA2-silenced cells relative to that of matching non-silenced controls. ( C , D ) The indicated cells were serum-starved for 24 h and seeded into Matrigel-coated transwell inserts (2 × 10 4 cells per well) in serum-free media ( n = 3 per group). The lower chamber contained media with 10% FBS. After 48 h, the number of invaded cells at the lower surface of insert porous membranes were quantified and graphed as in ( B ). ( E ) Migration assays were conducted using canine Eva and human MG63 (3 × 10 4 cells per insert) OS cells as in ( B ). Soluble ephrinA1 ligand or IgG (1 µg/mL) was added to the upper chamber at cell seeding. Graphs summarize data from two independent experiments and represent the percentage of migrated cells relative to IgG controls. Data are shown as mean ± SD. Experiments were repeated three times unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Cell Culture, Transferring, Imaging, Inverted Microscopy, Incubation, Migration, Membrane, Staining, Cell Counting

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 increases resistance to cisplatin. ( A ) The indicated canine OS cells were seeded into 96-well plates (4 × 10 3 cells per well) and allowed to adhere to the wells overnight. Culture media was replaced the next day with media containing increasing concentrations of cisplatin or media with PBS (as solvent control) at the volume matching the highest cisplatin dose as indicated ( n = 3 per group). The treated cells were cultured for 48 h. To assess cell survival, resazurin was added to the wells, and the fluorescence signal was quantified as in C. ( B ) The indicated human OS cells were tested for cisplatin sensitivity as described in A. Graphs represent cell survival as percentages relative to matching solvent controls. Data are shown as mean ± SD. Experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Solvent, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects osteosarcoma cell morphology. ( A , B ) The indicated canine and human OS cells were seeded into 6-well plates (50 cells per well, n = 3 per group) and allowed to grow into colonies for 7 days. Colonies were fixed with methanol and stained with crystal violet. The number of clustered, partially clustered, and spread colonies in each well were counted, and representative images were captured using a light microscope at 40× magnification. Graphs represent the proportion of each category of colonies relative to the total number of colonies. Representative images of these colonies are shown. ( C ) 143B-NS and 143B-shA2 cells were grown on glass coverslips to 50% confluency, fixed with 4% paraformaldehyde, and stained with rhodamine phalloidin (actin cytoskeleton, red) and DAPI (cell nucleus, blue). Fluorescent images were taken using an Olympus IX83 microscope at 200× magnification. Scale bar, 100 µm. Data are shown as mean ± SD. Experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Staining, Light Microscopy, Microscopy

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 promotes osteosarcoma tumor growth. ( A ) Abrams-NS and Abrams-shA2 osteosarcoma cells were injected subcutaneously in the flank area of 4–8-week-old male immunodeficient NOD–SCID gamma mice (1.0 × 10 6 cells per mouse in 100 µL PBS, n = 4 per group). Tumor growth was monitored every 3–4 days, and tumor volume was calculated as a 2 × b/2, where a and b are the short and long diameters, respectively. The graph represents a summary of two independent experiments. ( B ) Eva-NS and Eva-shA2 cells were injected and tumor growth was monitored as in A ( n = 6 per group). The graph represents one of two independent experiments. Data are shown as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Injection

Journal: Cells

Article Title: The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells

doi: 10.3390/cells13141201

Figure Lengend Snippet: EphA2 affects multiple signaling pathways in human and canine osteosarcoma cells. The indicated cells were cultured to approximately 70% confluency, serum-starved overnight, and the phosphorylation status of various proteins, as well as the expression of N-cadherin and Integrin β3, was assessed by Western blotting.

Article Snippet: The primary antibody against EphA2 (#12927, Cell Signaling Technologies) was diluted in PBS containing 1% BSA and 0.3% Triton X-100 and added to sections, followed by incubation with the antibody for 1 h at room temperature.

Techniques: Protein-Protein interactions, Cell Culture, Phospho-proteomics, Expressing, Western Blot

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 1. Eph proteins are sensitive to Hsp90 inhibition. A. PC3 and U251 cells were seeded in six-well plates and treated with 1 μmol/L GA for the indicated times. Cells were then lysed and equivalent protein subjected to SDS-PAGE and immunoblot analysis with EphA2 antibody. Tubulin was used as control for protein loading. B. PC3 cells were treated with 1 μmol/L GA, lysed as in A, and blots were probed for EphB2. C. HEK293 cells were transfected with plasmids encoding either HA-tagged EphA2 or EphB1. Cells were treated with 1 μmol/L GA for the indicated times, and drug concentrations and lysates were probed with anti-HA antibody. For the dose response, cells were treated for 16 h. Band intensities were quantitated with NIH Image J software and normalized to tubulin levels. The receptor levels from untreated cells were considered 100%, and the percentage of remaining receptor from drug-treated cells was determined relative to the control expression.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Inhibition, SDS Page, Western Blot, Control, Transfection, Software, Expressing

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 2. The kinase domain of EphA2 is required for chaperone recruitment and drug sensitivity. A. HEK293 cells were cultured in 10-cm dishes and transfected with 5 μg of the indicated plasmids. Following lysis, 1 mg total lysate was immunoprecipitated with HA-conjugated protein G beads and the blots probed for the indicated proteins. B. PC3 cells were treated with 1 μmol/L GA for the indicated times, EphA2 was immunoprecipitated as in A, and resultant blots were incubated with antibodies to either Hsp70 or EphA2. C. HEK293 cells were transfected with either full-length or kinase-deleted HA-tagged EphA2, treated for 16 h with GA, and equivalent protein was immunoblotted with HA antibody. D. Left, HEK293 cells were transfected as in C, treated with GA for 8 h, and 1 mg total protein was immunoprecipiated as in A. Resultant blots were probed for CHIP and EphA2 expression (anti-HA). Right, HEK293 cells were transfected with either pcDNA3.1 control vector, wild-type CHIP, or dominant-negative CHIP, and EphA2 levels were immunodetected as indicated.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Cell Culture, Transfection, Lysis, Immunoprecipitation, Incubation, Expressing, Control, Plasmid Preparation, Dominant Negative Mutation

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 3. Hsp90 inhibition facilitates EphA2 ubiquitination and degradation. A. Left, HEK293 cells were transfected with 5 μg HA tagged EphA2 and treated for 6 h with 1 μmol/L GA and/or 1 μmol/L proteasomal inhibitor PS-341 for the last 3 h. Following lysis, 1 mg of total protein was immunoprecipitated with anti-HA antibody and ubiquitinylated protein was immunodetected. The blot was then stripped and reprobed with anti-HA antibody for analysis of total EphA2 levels. Right, HEK293 cells were similarly transfected and treated with GA alone or with PS341, and EphA2 levels were detected from insoluble cell pellets following centrifugation and from soluble protein lysate. B. Logarithmically growing PC3 cells were starved in methionine-free medium 1 h, pulse labeled for 1 h in the presence of 1 μmol/L GA, and then chased for the indicated times in nonradioactive complete medium either lacking or containing 1 μmol/L GA. Endogenous EphA2 was immunoprecipitated from equivalent protein, and resultant gels were exposed to film and subject to densitometric analysis.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Inhibition, Ubiquitin Proteomics, Transfection, Lysis, Immunoprecipitation, Centrifugation, Labeling

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 4. Hsp90 inhibition impairs EphA2 lo- calization but not ligand binding and internaliza- tion. A. PC3 cells were cultured on fibronectin and treated with GA for the indicated times. The cells were then fixed and prepared for EphA2 im- munofluorescence staining. The numbers corre- spond to the percentage of cells exhibiting EphA2 localization at cell-cell contacts relative to untreated cells, as calculated from the average of four fields per coverslip, in duplicate. B. PC3 cells were pretreated with GA for the indicated times and then stimulated by A1-Fc (1 μg/mL) 30 min before fixation. C. PC3 and U251 cells were either untreated or treated for 8 h with 1 μmol/L GA and subsequently processed for flow cytometry as de- scribed. Values represent percentage of cells ex- pressing surface expression of EphA2. Representative data from duplicate experiments is shown.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Inhibition, Ligand Binding Assay, Cell Culture, Staining, Flow Cytometry, Expressing

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 5. Hsp90 regulates EphA2 receptor dimerization and phosphorylation. A. Top, PC3 cells were treated with GA for the indicated times, EphA2 was immunoprecipitated from 1 mg cell lysate, and resultant blots were probed for phosphotyrosine and reprobed for EphA2 to assess total receptor levels. Bottom, indicated cell lines were treated with GA for 9 h, EphA2 was immunoprecipitated from 500 μg cell lysate, and resultant blots were probed for phos- photyrosine. The percentage decrease in phosphorylation was calculated from the quantitation of receptor phosphorylation (ImageJ), normalized to total receptor levels. B. HEK 293 cells were cotransfected with myc-EphA2 and HA-EphA2 plasmids, treated as indicated, and HA-EphA2 was immunoprecipi- tated from lysates. Receptor dimerization was determined by visualization of myc-EphA2 copurifying with anti-HA immunoprecipitates. A control Myc immu- noprecipiation was also performed from myc–EphA2–transfected cells to validate signal specificity. Total levels of immunoprecipitated EphA2 were detected by anti–HA immunoblotting. C. To assess whether GA affects ligand-dependent cell rounding, cells were grown on fibronectin-coated cover slips and treated with A1-Fc for 10 min in either the presence or absence of pretreatment with GA. The cells were then fixed with paraformaldehyde and prepared for viewing. The percentage of rounded cells in response to ligand was determined from four fields, in duplicate, relative to the total number of cells per field, and nor- malized to the percentage of rounded cells in untreated samples.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Phospho-proteomics, Immunoprecipitation, Quantitation Assay, Control, Transfection, Western Blot

Journal: Molecular Cancer Research

Article Title: Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

doi: 10.1158/1541-7786.mcr-08-0582

Figure Lengend Snippet: FIGURE 6. EphA2 impairment potently inhibits cell migration to an extent similar to GA (top). U251 cells were pretreated with EphA2 antibody (10 μg/mL) or GA (1 μmol/L) 8 h before plating in Boyden chambers (ligand was added at time of plating), and cells were transfected with siRNAs 48 h before plating. Following a 16-h incubation period, migrating cells were counted as described and normalized to the number of migrating cells from DMSO-treated cells. Values corresponding to the normalized mean were converted to percent inhibition and subjected to statistical analysis. Representative siRNA-mediated knockdown of EphA2 is shown. PC3 cells (bottom) were treated as above, and the percent migration was similarly calculated.

Article Snippet: Cells were trypsinized, washed with PBS, blocked (0.1% sodium azide, 2% bovine serum albumin/PBS), and incubated with goat polyclonal EphA2 antibody (Santa Cruz) diluted 1:50 in blocking buffer.

Techniques: Migration, Transfection, Incubation, Inhibition, Knockdown

Journal: Nature methods

Article Title: Spatial control of membrane receptor function using ligand nanocalipers.

doi: 10.1038/nmeth.3025

Figure Lengend Snippet: Figure 2 | Ephrin-A5 conjugate and ephrin-A5–nanocaliper binding abilities. (a) Binding curves of ephrin-A5 conjugates (gray) and ephrin-A5 nanocalipers (green, light blue and dark blue) binding to the extracellular domain of human EphA2 measured by SPR. NC empty (purple) was used as a negative control. Inset, the apparent dissociation constants (Kdapp (nM), the binding curves were fitted with a 1:1 Langmuir binding model; Supplementary Fig. 9). RU, resonance unit. (b) FACS analysis of MDA-MB-231 cells stimulated for 15 min with IgG-clustered ephrin-A5 or NC-sat. IgG and NC-empty were used as controls (raw FACS are in the Supplementary Data). DyLight 488-conjugated anti-human IgG was used to precluster the ephrin-A5 for 15 min. DyLight 488–conjugated anti- human IgG was added to the cell culture medium 15 min after EphA2 stimulation with NC-sat and NC-empty. The percentage of fluorescently labeled cells is indicated in the plots, calculated as average ± s.e.m. from 2 independent biological repeats. FSC-A, forward scatter; FITC-A, fluorescein isothiocyanate.

Article Snippet: The membrane was incubated with goat anti-EphA2 antibodies (R&D systems, AF3035) (1:2,000 diluted in TBST with 1% milk powder) overnight at 4 °C.

Techniques: Binding Assay, Negative Control, Cell Culture, Labeling

Journal: Nature methods

Article Title: Spatial control of membrane receptor function using ligand nanocalipers.

doi: 10.1038/nmeth.3025

Figure Lengend Snippet: Figure 4 | Ephrin-A5 nanocalipers modulate EphA2 mediated responses in human breast cancer cells. (a–d) Immunocytochemistry of MDA-MB-231 cells using an antibody against the EphA2 receptor and the endosomal marker early endosome antigen (EEA1). Cells were stimulated for 120 min with IgG, ephrin-A5 monomer, ephrin-A5 dimer or IgG-clustered ephrin-A5 (a,b) or with NC-empty, NC0, NC100 or NC40 (c,d). Cells in b,d were treated with ephrin-A5 in the presence of Dyngo-4a, a dynamin inhibitor. (e) Relative fold decrease in matrix invasion of the breast cancer cells MDA-MB-231 after treatment with NC-empty, NC100 or NC40; n = 2. One-way analysis of variance (ANOVA) was used followed by Tukey’s multiple-comparison test (**P < 0.01, ***P < 0.001). Scale bars, 10 µm.

Article Snippet: The membrane was incubated with goat anti-EphA2 antibodies (R&D systems, AF3035) (1:2,000 diluted in TBST with 1% milk powder) overnight at 4 °C.

Techniques: Immunocytochemistry, Marker, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 1. EphA2 and EphA4 expression in 68 human cancer cell lines and 17 normal tissue RNA samples. (A) EphA2 and EphA4 receptor expression in normal and cancer cell lines. Bar graph shows the fold expression of both EphA2 and EphA4 receptors in MDA-MD-231, PC-3, and LNCaP cancer cell lines compared to cultured human dermal fibroblast (HDF) and to a total human prostate RNA sample (prostate). (B) Correlation between EphA2 mRNA expression and EphA2 protein levels in 30 selected human cancer cell lines. (C) Box and Whisker plots of the 10th to 90th percentile of EphA2 expression for the RNA samples grouped according to their tissue of origin. Dotted lines depict cutoffs for 7-fold and 14-fold expression levels observed in the reference HDF cell line, respectively.

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Whisker Assay

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 2. YSA peptide targets cells expressing EphA2. (A) The YSA peptide targets quantum dots to EphA2 on the cell surface. COS cells transfected with either the extracellular and transmembrane portions of EphA2 fused to enhanced green fluorescent protein (EphA2-EGFP) or membrane targeted EGFP-F were incubated with YSA bound to red fluorescent quantum dots (YSA−Qdots). YSA−Qdots only bind to cells transfected with EphA2-EGFP. EGFP fluorescence is green. Nuclei are stained in blue with DAPI. (B) YSA targets quantum dots to endogenous EphA2 on the cell surface. HUVE cells were incubated at 4 °C with YSA conjugated Qdots (YSA−Qdots), an unrelated 12- mer control peptide that does not bind to EphA2 also conjugated to Qdots (Ctrl-Qdots), or unconjugated Qdots (Qdots). Only quantum dots conjugated to the YSA peptide bind to the HUVE cells. (C) YSA targets Qdots to lysosomes. MDA-MB-231 cells, which express high levels of endogenous EphA2, were incubated at 4 °C with YSA−Qdots followed by incubation at 37 °C for 0, 15, and 60 min. YSA−Qdots are seen on the cell surface at 0 min but become concentrated in structures near the nucleus at 15 and 60 min (arrow). Double labeling with the lysosomal marker Lamp1 (green) shows colocalization of the quantum dots in lysosomes at 60 min (arrows).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Transfection, Membrane, Incubation, Fluorescence, Staining, Control, Labeling, Marker

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 3. YSA−PTX retains high potency for inhibition of EphA2-ephrin-A5 binding and high EphA2 binding affinity. (A) General chemical structures of YSA and DYP peptide−drug conjugates. (B) YSA, YSA−PTX, and control DYP−PTX were incubated at the indicated concentrations together with a constant concentration of ephrin-A5 AP in ELISA wells precoated with EphA2 Fc. The ratio of ephrin-A5 AP bound in the presence and in the absence of peptide is shown. (C) Biotinylated YSA−PTX and control DYP−PTX were incubated at the indicated concentrations in EphA2 Fc-coated ELISA wells and were then detected with streptavidin−HRP. The graphs show averages ± SE from quadruplicate measurements in representative experiments, while the KD and IC50 values are calculated from three to five experiments. (D) ATP-Lite analysis of viability of PC-3 cells 72 h after the addition of DYP, DYP−PTX, YSA, YSA−PTX, or PTX at 10 nM (n = 3).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Binding Assay, Control, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 4. YSA−PTX is internalized in cancer and endothelial cells expressing EphA2. (A) YSA−PTX coupled to fluorescent quantum dots, but not DYP−PTX, is internalized with EphA2 into lysosomes of prostate cancer cells expressing EphA2. PC3 prostate cancer cells, which express high levels of EphA2, or LNCaP prostate cancer cells, which do not detectably express EphA2 protein, were treated for 20 min with 100 μM YSA−PTX or control DYP−PTX, followed by a 20 min of incubation with streptavidin-conjugated quantum dots (Qdots). After removal of the solution containing the peptides and the quantum dots, the cells were incubated for 2 h at 37 °C to allow EphA2 internalization induced by YSA−PTX binding. The cells were stained for EphA2 (left) or the lysosomal marker Lamp1 (right). Qdots were also imaged, and nuclei were labeled with DAPI in blue. Representative fluorescence micrographs are shown. Scale bar = 25 μM. (B) Ephrin-A1 Fc and YSA−PTX, but not DYP−PTX, cause EphA2 internalization into lysosomes. PC3 cells were treated for 2 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA−PTX, or 100 μM control DYP−PTX. The cells were stained for Lamp1 (red) and EphA2 (green). Nuclei were labeled with DAPI (blue). Representative confocal micrographs are shown. Scale bar = 25 μm. (C) Ephrin-A1 Fc and YSA−PTX, but not DYP−PTX, cause EphA2 internalization into cells. PC3 prostate cancer and HUVE endothelial cells were treated for 1 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA−PTX, or 100 μM control DYP−PTX. Proteins present on the cell surface were then labeled with biotin. EphA2 immunoprecipitates were probed with antiphosphotyrosine antibody (PTyr), streptavidin−HRP (biotin), and EphA2 antibody. The amount of GAPDH in the cell lysates used for the immune precipitations is also shown as a control for equal amounts of protein in the lysates used for immunoprecipitation.

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Incubation, Binding Assay, Staining, Marker, Labeling, Fluorescence, Immunoprecipitation

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 5. Expression of EphA2 and cellular and in vivo activity of drug conjugates. (A) Groups of five to seven SCID beige mice bearing pre- established subcutaneous PC3 tumors were treated with twice weekly with intravenous doses of vehicle (CT), paclitaxel (5 mg/kg), YSA− paclitaxel (equimolar doses of the paclitaxel dose), starting at day 0. Tumor sizes were measured, and averages ± SEM are shown. p < 0.05 for the comparison of YSA−paclitaxel with control, by repeated- measures two-way ANOVA using all the measurements as well as by one-way ANOVA and Dunnett’s post hoc test using the measurements at day 18. (B) Biodistribution analysis. PC3 tumors (n = 3, average volume of 200 mm3) were excised 30 min after equimolar amounts of YSA−PTX or PTX (25 mg/kg) were injected intravenously. LC−MS analysis was performed to quantify PTX levels from tumor extracts. The histogram shows averages ± SEM. p < 0.05 for the comparison of PTX levels, by t test analysis. Plasma levels of PTX were not significantly different (YSA−PTX = 998 ± 742 ng/mL; PTX = 771 ± 585 ng/mL).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, In Vivo, Activity Assay, Comparison, Control, Injection, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a FGF4 is a key signal that promotes differentiation of pluripotent mESCs. b Workflow for phosphoproteomic analysis of FGF4 signalling in Fgf4 −/− mESCs. Volcano plot showing significantly modified phosphosites after stimulation of Fgf4 −/− mESCs with FGF4 for 5 min ( c ) and 20 min ( d ). Phosphosites on known FGF4 pathway components are highlighted. e Protein kinase phosphopeptides that are significantly upregulated (>2-fold) on at least one time point (5 or 20 min) compared with control. Data are presented as mean ± SD ( n = 3). f Fgf4 −/− mESCs were stimulated with FGF4 for the indicated time, and EPHA2 pS898 and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Modification, Control, Western Blot

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Average protein copy number per cell determined for receptor kinases in mESCs, using quantitative whole-cell proteomics. Data are presented as mean ± SD ( n = 3). b Workflow for quantification of EPH–EFN interactions in mESCs by EFN ligand affinity purification mass spectrometry. c Proof-of-principle identification of EPH–EFN interactions by EFN ligand affinity purification. EPHA2 levels were determined by immunoblotting. d Coomassie staining of EFNA1/B1 affinity purification from mESCs. EFNA1, EFNB1 and EPHA2 proteins are indicated. (*) = non-specific band. e Mass-spectrometry analysis of 75–130-kDa region of the Coomassie stained EFNA1/EFNB1 affinity purification shown in ( d ). Total spectral counts recovered for each EPH receptor family member are indicated. f EFNA1/B1 affinity purification from intact Epha2 +/+ and pooled Epha2 −/− mESCs. Phosphotyrosine (pTyr), EPHA2 and ERK1/2 levels were determined by immunoblotting. Note that the pTyr signal is specific for EPHA2, and is not detected in the absence of EFN ligand. g EPHA2 was immunoprecipitated from Epha2 +/+ and Epha2 −/− mESCs, and pTyr, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting. h Epha2 +/+ mESCs were stimulated with EFNA1-expressing Epha2 −/− mESCs for 15 or 40 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Affinity Purification, Mass Spectrometry, Western Blot, Staining, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs (clone C4) stably expressing EPHA2 were cultured in the absence of LIF for 48 h. EPHA2, KLF4, DNMT3B, NANOG and OCT4 levels were determined by immunoblotting. b Epha2 +/+ or Epha2 −/− (clone C4) mESCs were maintained in 2i or differentiated in N2B27 media for 72 or 96 h, respectively, whereupon 10% of cells were replated in 2i. Total alkaline phosphatase staining is represented relative to Epha2 +/+ mESCs. The total number of alkaline phosphatase-positive colonies for Epha2 +/+ and Epha2 −/− mESCs is shown, and also represented relative to Epha2 +/+ mESCs. Data show mean ± SEM ( n = 3); statistical significance was determined using unpaired two-sided Student’s t test comparing Epha2 −/− with the Epha2 +/+ control (**** P < 0.0001, ** P = 0.0016). c Epha2 +/+ or Epha2 −/− (clone C4) mESCs stably expressing EFNA1, along with the respective parental controls, were grown in LIF/FBS, and KLF4, NANOG, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting, or EPHA2 immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. d Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs stably expressing EPHA2 were differentiated as embryoid bodies for 10 days, and the levels of Fgf5 , Brachyury , Mixl and Cer1 mRNA determined by qRT-PCR. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. The results shown are for technical replicates from two independent experiments, including three Epha2 −/− clones ( n = 3); statistical significance at day 4 was determined using unpaired two-sided Student’s t test comparing each group with the Epha2 +/+ control (ns = not significant, * P = 0.0252, ** P = 0.0045, *** P < 0.0001). e Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. ppERK1/2, total ERK1/2, STAT3 pY705 and total STAT3 levels were determined by immunoblotting. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. ppERK1/2 signal was quantified; data show mean ± SD ( n = 3); statistical significance was determined using one-sample two-sided t test comparing each group with control, theoretical mean = 1 (ns = not significant, 5 min; * P = 0.0467, 30 min; *** P = 0.0005, 45 min; ** P = 0.0014, 60 min; ** P = 0.0018). f Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. SHP2 was immunoprecipitated, and SHP2 and EPHA2 levels detected by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Staining, Control, Immunoprecipitation, Quantitative RT-PCR, Whisker Assay, Clone Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Diagram of potential phosphorylation sites within the EPHA2 S898 motif. Mass spectrometry analysis detects phosphorylation of at least three sites in EPHA2 immunoprecipitated from FGF4-stimulated Fgf4 −/− mESCs (see Supplementary Table ). b Fgf4 −/− mESCs were treated with 10 μM of the indicated inhibitors for 1 h, and stimulated with FGF4 for 10 min. EPHA2 pS898, EPHA2 and AKT pS473 levels were determined by immunoblotting. c Epha2 −/− mESCs were transfected with either wild type or 5E EPHA2 constructs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting and quantified. Data show mean ± SD ( n = 4). d EPHA2 was immunoprecipitated from EPHA2 WT knock-in (KI) or 5A KI cell lines and pTyr and EPHA2 levels determined by immunoblotting (upper panel). Relative pTyr/EPHA2 signal was quantified (lower panel). Data show mean ± SD ( n = 4). e Phase-contrast images of EPHA2 WT KI or 5A KI mESC lines; scale bar = 100 µM. f EPHA2 WT KI or 5A KI cell lines were cultured in LIF/FBS medium for 48 h, and KLF4, NANOG, DNMT3B, OCT4 ppERK1/2 and ERK1/2 levels determined by immunoblotting. g Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EPH receptor expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). h Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EFN ligand expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). Epha2 ( i ) and Efna1 ( j ) mRNA expression in 2i mESCs undergoing differentiation in N2B27 was determined by qRT-PCR analysis at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of two technical and three biological replicates ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Phospho-proteomics, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Knock-In, Cell Culture, Expressing, Quantitative RT-PCR, Whisker Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ mESCs were transfected with the indicated siRNAs, and EPHA2, OCT4 and SOX2 levels determined by immunoblotting. A non-specific band was used as a loading control. b Epha2 and Oct4 mRNA expression was determined by qRT-PCR following transfection of Epha2 +/+ mESCs with control or OCT4 siRNA. Data show mean ± SEM of four technical replicates (n = 4). c Pluripotency transcription factor-binding sites in Epha2 gene regulatory regions were extracted from CODEX mESC ChIP-SEQ data ( http://codex.stemcells.cam.ac.uk ). d Epha2 +/+ mESCs were transfected with the indicated siRNAs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated using EPHA2 antibody, and pTyr and EPHA2 levels determined by immunoblotting. e EFNA1/B1 affinity purification from control, dasatinib-treated or siOct4-transfected mESCs. pTyr, EPHA2, OCT4 and ERK1/2 levels were determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Transfection, Western Blot, Control, Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Immunoprecipitation, Affinity Purification

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: Expression (RPKM) of EPH ( a , b ) or EFN ( c , d ) mRNA in the inner cell mass (ICM) or trophectoderm (TE) of 64-cell mouse embryos ( a , c ) or 5-day human embryos ( b , d ). a , c n = 33 biologically independent cells for ICM, and n = 28 biologically independent cells for TE. b , d n = 73 biologically independent cells for ICM, and n = 142 biologically independent cells for TE. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. e EPHA2 regulation and function in mESCs. In the pluripotent state, OCT4 and other pluripotency factors promote EPHA2 receptor expression, enabling activation by EFNA ligands to support pluripotency by restraining ERK1/2. During differentiation, FGF4 drives ERK1/2–RSK activity to phosphorylate and inhibit EPHA2, whilst ERK1/2 suppresses an OCT4–EPHA2 transcriptional module to disable EPHA2 receptor expression. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Expressing, Whisker Assay, Activation Assay, Activity Assay

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) The EphA2 transcript was repressed by siRNA in brain endothelial cells. Quantitative PCR revealed ~51% repression of EphA2 transcript in contrast to the siRNA control. (B) Transcytosis assays in the in vitro BBB model show a significant reduction in the movement of C. neoformans across endothelial cells when EphA2 transcript is repressed. Transmigration of C. neoformans was not affected when endothelial cells were transformed with an siRNA negative control. Transcytosis assays were performed as described below. Fungal cells were collected from bottom well following 24h co-incubation (n=5, *P<0.05). (C) Following siRNA of EphA2 transendothelial electrical resistance measurements (TEERs) were performed at the indicated times with an endometer. The TEER measurements indicate an intact barrier. The modest upward trend of TEERs suggests a tightening of the barrier. (Lower left panel) A schematic diagram depicting an in vitro, static monolayer model of the human BBB. Immortalized human brain endothelial cells (hCMEC/D3 cell line) are grown and differentiated on a transwell. Barrier integrity is confirmed by monitoring tightness of barrier junctions with TEERs and dextran permeability. Transcytosis assays are performed as follows: Fungal cells are added to the luminal side (top of transwell), collected from the bottom well (abluminal side of BBB) at indicated times and plated onto agar plates for CFU determination.

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Control, In Vitro, Transmigration Assay, Transformation Assay, Negative Control, Incubation, Permeability

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) Addition of peptide monoclonal antibody (mAb) against the extracellular N-terminal region of EphA2 blocks activity of EphA2. Brain endothelial cells were treated with EphA2-mAb (125μg) for 45mins and subsequently challenged with C. neoformans. Following a 3h co-incubation in the in vitro BBB model, transcytosis assays showed reduced fungal crossing, in contrast to the control antibody (IgG). (B) Treatment of endothelial cells with the antibodies did not affect dextran permeability suggesting an intact barrier. (C) & (D) Similarly, addition of 10μm or 20μm dasatinib (an inhibitor of EphA2) to brain endothelial cells in the in vitro BBB model, reduced fungal crossing but did not appear to alter dextran permeability. The DMSO solvent control had no effect on fungal crossing. Transcytosis assays were performed as described above. n=8, *P<0.05, ***P<0.001, n/s = not significant.

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Activity Assay, Incubation, In Vitro, Control, Permeability, Solvent

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) Western blot analysis demonstrated the phosphorylation of EphA2 in brain endothelial cells when cells were challenged with C. neoformans (B) S. cerevisiae or (E) cps1Δ for 15min, 30min or 1h (middle panel). A polyclonal anti-phospho antibody to EphA2 was used to detect the phosphorylated form of EphA2. GAPDH was used as a loading control. (C) Endothelial cells treated with dasatinib and co-incubated with C. neoformans revealed a lack of EphA2 phosphorylation, however DMSO control clearly indicated phosphorylated EphA2, thus consistent with the notion that C. neoformans activates EphA2. (D, F) Relative band intensity of phosphorylated EphA2.

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Western Blot, Phospho-proteomics, Control, Incubation

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) Addition of human recombinant ephrinA1 (EFNA1, Origene), a ligand of EphA2, to brain endothelial cells enhanced crossing of C. neoformans in the in vitro BBB model. Three concentrations of EFNA1 (1, 1.5, and 2 μg/ml) were co-incubated with 2×105 cells of C. neoformans cells and added to the top chamber of the transwell in the in vitro BBB model. Following 24h, fungal cells were collected from the bottom chamber and placed onto agar plates for CFU determination. (B) Transcytosis assay revealed that the EphA2 agonist, doxazosin, facilitated migration of C. neoformans across brain endothelial cells. DMSO, a solvent for doxazosin and used here as a negative control, had no effect. Brain endothelial cells were exposed to 100μM doxazosin and co-incubated with 2×105 cells of C. neoformans. Following 24h, fungal cells were collected from bottom chamber placed onto agar plates for CFU determination *P<0.05, n=8.

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Recombinant, In Vitro, Incubation, Migration, Solvent, Negative Control

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: The EphA2 receptor co-localized with F-actin and both surrounded C. neoformans (indicated by arrows). In addition, clustering of the EphA2 receptor was observed on adjacent brain endothelial cells in close proximity to fungal cells (indicated by star). Panels (A) & (B) represent merged confocal images of immunofluorescence of endothelial cells exposed to C. neoformans; (C) F-actin was detected by phalloidin (yellow); (D) The EphA2 receptor is shown in red, (E) nuclei are shown in blue with DAPI stain and (F) C. neoformans was detected by FITC (shown in green).

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Immunofluorescence, Staining

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: Confocal microscopy revealed that brain endothelial cells exposed to C. neoformans produced microvilli-like structures of F-actin that embraced C. neoformans. EphA2 displayed a punctate localization pattern and co-localized with F-actin in association with C. neoformans (indicated by arrows). As noted above, a cluster of EphA2 receptors was clearly observed on the surface of brain endothelial cells that were very near to fungal cells (indicated by star). Panels (A) & (B) represent merged confocal images of immunofluorescence of endothelial cells exposed to C. neoformans. (C) Illustrates F-actin as detected by phalloidin (yellow); (D) The EphA2 receptor is shown in red; (E) nuclei are shown with DAPI stain (blue) and (F) C. neoformans was detected by FITC (shown in green).

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Confocal Microscopy, Produced, Immunofluorescence, Staining

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A)–(E) Immunofluorescence images represent still images captured at indicated time points from live-cell imaging of HEK293T cells expressing EphA2-cDNA and challenged with C. neoformans (S6_movie & S7_movie). Images display a clear association between EphA2 and C. neoformans as indicated by cell protrusions engulfing fungal cells (white arrows). Fungal cells were stained with FITC (green); Expressed EphA2 in HEK293T cells is shown in red; Nuclei are stained with DAPI and appear blue. (F) Western blot analysis detected the presence of the EphA2 in HEK293T cells (indicated by black arrow). The polypeptide band corresponding to EphA2HIS was not observed in non-transfected cells. GAPDH was used as a loading control (indicated by bottom black arrow).

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Immunofluorescence, Live Cell Imaging, Expressing, Staining, Western Blot, Transfection, Control

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) To establish whether EphA2 acted directly to internalize C. neoformans a cell protection assay was performed. HEK293T cells overexpressing EphA2 were exposed to C. neoformans for 1.5h, subsequently washed away and replaced with fluconazole (15μg/ml), a static antifungal drug. Following a further 48h co-incubation where internalized C. neoformans was protected from fluconazole and allowed to replicate, HEK293T cells were lysed and plated for CFU determination (B) Significantly more CFUs from HEK293T overexpressing EphA2 than HEK293T cells alone (transformed with an empty plasmid) were detected, suggesting that EphA2 was directly responsible for the internalization of C. neoformans. (C & D) Prior to the assay, fluconazole activity was monitored to ensure HEK293T cells remained viable and fungal cells were susceptible.

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Incubation, Transformation Assay, Plasmid Preparation, Activity Assay

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: (A) Addition of human recombinant ephrinA1 (EFNA1, Origene) to brain endothelial cells did not rescue the crossing defect of the C. neoformans cps1Δ deletion strain. Two concentrations of EFNA1 (1 and 2 μg/mL) were co-incubated with 2×105 cells of C. neoformans cells and added to the top chamber of the transwell in the in vitro BBB model. Following 6h co-incubation, fungal cells were collected from the bottom chamber and placed onto agar plates for CFU determination, n=8 (B) Transcytosis assays with the EphA2 agonist, doxazosin, did not enhance crossing of the cps1Δ strain. DMSO is the solvent control. Brain endothelial cells were exposed to 100μM doxazosin and co-incubated with 2×105 cells of C. neoformans. Following 6h, fungal cells were collected from bottom chamber placed onto agar plates for CFU determination, n=5. (C) Brain endothelial cells were treated with EphA2-mAb (125μg) for 45mins and subsequently challenged with C. neoformans. Following a 3h co-incubation in the in vitro BBB model, transcytosis assays showed reduced crossing of WT and cps1Δ cryptococci, n= 8. (D) Addition of 10μm dasatinib to brain endothelial cells in the in vitro BBB model reduced crossing of WT and cps1Δ cryptococci, n= 5. (F, G, H) Barrier integrity was monitored by dextran permeability during treatments of brain endothelial cells with the antibodies (E) The defect in the transmigration of the cps1Δ strain is not due to growth inhibition in the endothelial cell culture medium. *P<0.05, **P<0.01, ***P<0.0001, n/s = not significant

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Recombinant, Incubation, In Vitro, Solvent, Control, Permeability, Transmigration Assay, Inhibition, Cell Culture

Journal: Cellular microbiology

Article Title: The Human Blood-Brain Barrier Internalizes Cryptococcus neoformans via the EphA2-Tyrosine Kinase Receptor

doi: 10.1111/cmi.12811

Figure Lengend Snippet: C. neoformans associates directly with the luminal side of the brain endothelium and induces EphA2 phosphorylation through transactivation of CD44 bound to C. neoformans. This promotes GTPase-dependent signaling that reorganizes the actin cytoskeleton and internalizes C. neoformans via endocytotic and transcellular mechanisms that require Mpr1 and Annexin A2 (AnxA2).[18,69] Sustained EphA2 activation could weaken intercellular junctions thereby increase paracellular permeability and boost further entry of C. neoformans along with excess fluid that would lead to brain edema (indicated by dashed arrows).

Article Snippet: The PVDF membranes were then incubated with primary antibodies (Rabbit polyclonal EphA2 IgG (Santa Cruz Technology) or phospho-EphA2 S897 (Cell Signaling) or histidine-6 (Neuromab, Inc) diluted at 1:1000 overnight at 4°C.

Techniques: Phospho-proteomics, Activation Assay, Permeability

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Top, partial amino acid sequence of the human EphA2 receptor showing the TM helix (underlined), preceded by a short extracellular segment, and followed by the start of the juxtamembrane segment. Residue numbers in the sequence of EphA2 are shown. Middle , sequence of the TYPE7 peptide, where the acidic residues introduced are shown in red. Bottom , sequence of the TMJM 563 -EphA2 peptide used in panel D. ( B ) Circular dichroism determination of TYPE7 secondary structure in buffer at pH 8 (grey line), and in the presence of POPC vesicles at pH 8 (dotted blue line) and after acidification to pH 4 (red line). ( C ) TYPE7 binding to POPC vesicles at pH 5 (red) and pH 8 (blue). Lines are fittings to , used to determine the Kp values. Lipid binding was measured using the environmentally-sensitive dye NBD attached to the N t of TYPE7. ( D ) Determination of the pH midpoint (pH 50 ) for the insertion of TYPE7 into POPC vesicles. TYPE7 data is shown in red symbols. Data obtained in vesicles containing the GWALP23 peptide control are shown in grey, and in vesicles containing TMJM 563 -EphA2 in orange. Peptide insertion was monitored by following changes in the NBD spectral center of mass ( ; ). Control OCD experiments showed that TMJM 563 -EphA2 formed a TM helix . The lines correspond to the fitting to the data using and 95% confidence intervals are shown as shaded areas ( n = 6). ( E ) OCD determination of the membrane orientation of TYPE7. Data were obtained in POPC (16:0,18:1-PC, dashed red line) and 22:1,22:1-PC (continuous red line). The theoretical spectra for a perfectly transmembrane (0°, black line) and peripheral (90°, grey line) helix are shown as a reference. ( F ) Cartoon of the different states TYPE7 (blue) adopts, and how TMJM 563 -EphA2 (orange) promotes the TM state of TYPE7. Arrows represent approximate equilibrium conditions found at pH ~6.5. The (+) symbols represent basic residues in the juxtamembrane segment of EphA2.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Sequencing, Residue, Circular Dichroism, Binding Assay, Control, Membrane

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) TYPE7-bodipy FL-X binding to H358 cells at pH 5, 6 and 7. Data at different pH values were normalized to maximum fluorescence. Mean ±S.D., n = 3. Student’s t -test; *p<0.05; **p<0.01 and NS: not significant. ( B ) H358 cells were treated with increasing concentrations of TYPE7 (0.5, 1 and 2 μM) during 24 hr. Cell viability was assessed using the MTS assay. The results indicate that TYPE7 does not cause toxicity to treated cells. Mean ±S.D., n = 3. ( C ) We threaded the sequence of TYPE7 (blue) onto one of the helices of the published dimeric structure of the transmembrane domain of EphA2 (PDB: 2K9Y) (orange). The residues substituted with glutamic acid are shown as spheres on TYPE7 outside the helix interface. The corresponding EphA2 residues are highlighted on the opposite orange helix.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Binding Assay, Fluorescence, MTS Assay, Sequencing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) OCD spectrum of TMJM 563 -EphA2 in POPC (16:0,18:1-PC) bilayers. ( B ) HPLC data showing that TMJM 563 -EphA2 does not dimerize using a disulfide bond. Top , chromatogram showing the elution of the TMJM 563 -EphA2 monomer at 26.2 min. Bottom , control experiment where TMJM 563 -EphA2 dimerization was induced by oxidation with 10 mM copper phenanthroline for 3.5 hr. A dimeric peak appears at 20.2 min, which was not observed in the absence of oxidizing agent.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Confocal microscopy shows co-localization of TYPE7 and EphA2. A375 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 0.2 µM TYPE7-Alexa568 (red) for 5 min at room temperature. Cells were fixed and endogenous EphA2 was labeled via immunofluorescence (green). Images were collected using a 63x objective, and insets show images corresponding to the white dashed areas collected with a 100x objective. Scale bars are 20 µm and 5 µm, respectively. ( B ) The Pearson correlation coefficient (r) was calculated for cells incubated with TYPE7 in the absence and presence of EA1. Bar graph shows mean ±S.D. Student’s t -test was performed for 14 – 17 images. *p<0.05, with as effect size of 0.80 standard deviations, n = 2. ( C ) Top, SDS-PAGE showing that TYPE7-DL co-precipitates with endogenous EphA2 when using a polyclonal anti-rabbit EphA2 antibody. Middle , control Western blots of EphA2 immunoprecipitation blotted with mouse anti-EphA2 show that similar amounts of endogenous EphA2 were pulled down in all samples. Total cell lysates blotted with EphA2 and β-actin indicate that similar levels of protein were loaded. Bottom, quantification of the fluorescent bands. Bar graph shows mean ±S.D. as a percentage of maximum intensity. A Mann-Whitney test was performed (*p<0.05), n = 3.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Confocal Microscopy, Incubation, Labeling, Immunofluorescence, SDS Page, Control, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Left , cell migration was measured in the presence and absence of TYPE7 and EA1 using a Boyden cell chamber assay. Representative images are shown. Right , quantification of migrating cells, showing that incubation with TYPE7 reduced A375 cell migration to a similar degree as EA1, with effect sizes of 8.4 and 12.6 standard deviations from control, respectively. N = 3. Cells were treated with an isolated Fc group as a control for the Fc present in EA1. Scale bar is 200 µm ( B–E ), Phosphorylation of Y772 and JMS phosphorylation at Y588 and Y594. A representative Western blot is shown ( B ). Band intensity was quantified for p-Y772 ( C ), p-Y588 ( D ), and p-Y594 ( E ). We found that incubation with TYPE7 increased phosphorylation of Y772 as efficiently as EA1, with effect sizes of 5.1 and 7.7 standard deviations from control, respectively. Mean ±S.D. are shown. n = 5. ( F–H ), Phosphorylation levels of Akt. A representative Western blot is shown ( F ) and band intensity was quantified for p-T308 ( G ) and p-S473 ( H ). Lysates were blotted against total EphA2 to detect total protein levels, and β-actin as a loading control. Student’s t -test was performed to obtain p values (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 and NS, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, Boyden Chamber Assay, Incubation, Control, Isolation, Phospho-proteomics, Western Blot

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Comparison of the sequences of TYPE7 and pHLIP, with acidic residues marked in orange. Experiments were performed in A375 cells ( B ) and H358 cells ( C ). Top panels , cell lysates were blotted with anti-phospho-EphA2 Y772, and EphA2 and anti-β-actin as loading controls; Bottom panels , quantification of p-EphA2 Y772 bands. Cells were treated with Fc, TYPE7 (2 μM), pHLIP (2 μM), or EA1 (0.5 μg/mL). Statistical analysis was performed using a Student’s t -test; *p<0.05, NS = no significant differences. n = 4 – 6 for panel B, and n = 3 for panel C. All experiments were performed at pH 7.4, except pHLIP in panel B, which was performed at pH 4.2 to ensure complete TM helix formation of pHLIP.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 μg/mL), TYPE7 (2 μM) or EA1 (0.5 μg/mL). The cell lysates were blotted with anti-phospho-EphA2 S897 and anti-β-actin to assess total protein loading. ( B ) EphA2-phospho-S897 quantification of five independent experiments. Statistical analysis was performed by using a Student’s t -test, which indicated no significant differences between samples and controls. ( C ) MTS cell proliferation assay. A375 cells were treated with Fc (0.5 μg/mL), EA1 (3 μg/mL), TYPE7 (2 μM) and TYPE7 +EA1 for 48 hr. No significant differences between Fc control and TYPE7 treated cells were found using a Student’s t -test; **p<0.01. Mean ±S.D., n = 3. ( D–E ) EphA2 expression levels do not change after TYPE7 treatment. Student’s t -test was performed and no significant differences were found between samples. Mean ±S.D., n = 5.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Proliferation Assay, Control, Expressing

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) H358 cells were treated with Fc (0.5 mg/mL), TYPE7 (2 μM), pHLIP (2 μM) or EA1 (0.5 mg/mL). After treatment, cell lysates were incubated overnight with array membranes to detect tyrosine phosphorylation of 49 different RTKs. The three pairs of reference spots used for blot alignment are boxed pink. Boxed RTK are: EphA1 (blue), EphA2 (yellow), HGFR/c-MET (green), EGFR (red) and ErbB3 (orange). ( B ) Bar graph shows mean and standard deviation of selected RTKs. The table on the right shows the identity of all the RTKs. ( C ) pHLIP weakly promotes phosphorylation of ErbB3 and HGFR/c-MET, as TYPE7 does. Since pHLIP does not induce EphA2 phosphorylation at Y772 or affects cell migration , this evidence logically argues against activation of those RTKs being involved in the TYPE7 regulation of these events.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Phospho-proteomics, Standard Deviation, Migration, Activation Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) Super-resolution SIM data. H358 cells were incubated in the presence (+) or absence (-) of 0.5 µg/mL EA1 and 2 µM TYPE7. Representative images show fluorescence obtained using an anti-EphA2 antibody ( n = 4). Scale bar is 10 µm. Insets magnify areas with clusters, and the scale bars are 5 µm. ( B ) Representative FCS autocorrelation curves for EphA2FL-GFP in control conditions (green) or in the presence of TYPE7 (blue) and EA1 (magenta). Δτ1 and Δτ2 represent the changes in dwell time. ( C–E ) Diffusion coefficient results, containing graphic models describing the EphA2 constructs used. ( C ) Box-whisker plot of measurement of the FCS diffusion coefficient of EphA2FL-GFP. ( D ) Diffusion coefficient of EphA2ΔJ-GFP. ( E ) Diffusion coefficient of Myr-EphA2 ICD-GFP. Diffusion coefficients collected from cells with and without TYPE7 treatment are reported along with EA1 ligand stimulation (orange boxes). The median values are reported next to the box plots. Each data point is the average of five 10 s FCS measurements on one cell. The grey numbers on top of the plots are the total number of cells measured. Criteria for the box, median, quartiles, whiskers and outliers are described elsewhere . One-way ANOVA tests were performed to obtain the p values (****p<0.0001; ns, not significant).

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Fluorescence, Control, Diffusion-based Assay, Construct, Whisker Assay

Journal: eLife

Article Title: A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

doi: 10.7554/eLife.36645

Figure Lengend Snippet: ( A ) FCS experiments. Schematic diagram of a FCS experiment. A 488 nm laser beam is focused at the peripheral membrane area of a cultured cell to excite the GFP tag on the diffusive receptors. The emitted photons are collected through the objective and directed to an avalanche photodiode (APD). The fluorescence fluctuation caused by the diffusion of receptors is recorded and transformed into the auto-correlation function. Insert: epi-fluorescence image of DU145 cell expressing GFP-tagged receptors; the red dot represents the position of laser beam. Scale bar is 5 μm. In the auto-correlation curve, τ D and G(0) report on the mobility and the concentration of the diffusive receptors, respectively. ( B ) FCS auto-correlation curves for the three EphA2 constructs. Three curves are shown for each experimental condition. ( C ) Receptor density of EphA2FL-GFP in DU145 cell membranes. Median density value is reported for EphA2FL-GFP and EphA2ΔJ-GFP. Each data point is the average of five 10 s FCS measurements on one cell. 52 cells were measured. ( D ) Representative epi-fluorescence images of cells used for FCS measurements under different conditions of TYPE7 and EA1 treatment. Scale bars are 5 μm.

Article Snippet: Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Membrane, Cell Culture, Fluorescence, Diffusion-based Assay, Transformation Assay, Expressing, Concentration Assay, Construct

Journal: Protein science : a publication of the Protein Society

Article Title: Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

doi: 10.1002/pro.4824

Figure Lengend Snippet: FIGURE 4 Analysis of S1-mediated crystal lattice packing interactions in FabS1CE-C1:-EPHA2-CRD and FabS1C-C1 structures. (a) Crystal lattice packing arrangement (upper panel) with symmetry mates, and asymmetric unit (lower panel) of (i) FabS1CE-C1: EPHA2-CRD (P3221 space group, trigonal crystal system), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group, monoclinic crystal system), and (iii) FabS1C-C1 (P21 space group). ASU Fab heavy- and light-chains are colored light blue or gray, respectively. Symmetry mate Fab heavy- and light-chains are colored dark blue and green, respectively. EPHA2-CRD is colored magenta. The heavy- and light-chain variable (VH and VL) and constant domains (CH and CL) of the Fab are labeled in (i). (b) S1 substitutions (Q165S and K167Y) and residues in the nearby loop region (N170, A171, L172. S174) cooperate to form crystal lattice packing sites in the following structures: (i) FabS1CE-C1: EPHA2-CRD (P3221 space group), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group), and (iii and iv) FabS1C-C1. NB: In the S1-crystal lattice packing site of FabS1CE-C1:EPHA2-CRD (P21 space group) (ii), the K72 side chain of the packing Fab VH domain remains partially unresolved from the electron density indicating a dynamic interaction.

Article Snippet: Cell culture expressing EPHA2-CRD or antigen-B was supplemented with 5 mM Kifunensine (MedChemExpress) to inhibit mannosidase I activity.

Techniques: Labeling

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: Figure 1. 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: Figure 3. 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: t0 = 0 min, t1 = 140 min, t2 = 280 min, t3 = 420 min, t4 = 560 min. For a-CD276: t0 = 0 min, t1 = 220 min, t2 = 440 min, t3 = 660 min, t4 = 880 min. For a-CD70: t0 = 0 min, t1 = 350 min, t2 = 700 min, t3 = 1050 min, t4 = 1400 min. N = 2 biological replicates

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: Figure 5. 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-Vhigh 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