pcmv6 epha2 gfp  (OriGene)

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 1. Sketch layout of PIE-FCCS and its role in membrane protien interaction. (A) Diagram illustrates the optical path of dual-color PIE-FCCS. A super-continuum, pulsed laser-generated excitation beams (488 and 561 nm) that were directed into the sample by a dichroic mirror and microscope objective. The lasers were focused to a diameter of about 250 nm on the plasma membrane of live cells. Fluorescence emission was collected by the objective, filtered, and directed to single-photon counting modules con- nected to a TCSPC module for data recording. (B) Epifluorescence images are shown for COS-7 cells expressing EGFR-mCH (top) and EphA2-eGFP (bottom). (C) Summary data from independent experiments collected on five different days of single-cell measurements are shown for the controls and EGFR with EphA2 (***P < 0.0004). (D to F) Repre- sentative single-cell PIE-FCCS data are shown for a monomer control (SRC), dimer control (GCN4), and EGFR with EphA2 in COS-7 cells, respectively. In each graph, the green line is the autocorrelation function (ACF) for eGFP, the red line is the ACF for mCH, and the blue line is the cross-correlation function (CCF) between both protein constructs.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Membrane, Generated, Microscopy, Clinical Proteomics, Fluorescence, Expressing, Control, Construct

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 2. PIE-FCCS investigation of the homomeric interactions of EGFR and EphA2. (A) Self-assembly of EGFR was studied under various conditions. PIE-FCCS data were recorded, and the resulting fc values are reported before treatment (white), after the addition of EGF (gray), after treatment with the PLC inhibitor U73122 (blue), and after treatment with the PLC activator 3M3FBS (green). (B) Diffusion coefficients for EGFR are shown for each treatment condition. (C) Epifluorescence images of EGFR express- ing COS-7 cells without (top) and with (bottom) EGF ligand treatment. (D) Homomultimerization state of EphA2 was recorded under the same treatments except the li- gand treatment was with EA1. (E) Diffusion coefficients are shown for EphA2 under each treatment condition. (F) Epifluorescence images of EphA2 expressing COS-7 cells are shown without (top) and with (bottom) EA1 ligand treatment. In the box-and-whiskers plots, the whiskers indicate the maximum and minimum values; the box indi- cates the 25th to 75th percentile, and the line is the median value (median value shown as text). We performed one-way analysis of variance (ANOVA) tests with uncor- rected Fisher’s least significant difference (LSD) post hoc tests to obtain adjusted and individual P values (*P < 0.0367, **P < 0.0034, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM. NS, not significant.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Diffusion-based Assay, Expressing

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 3. Heteromultimerization of EGFR and EphA2 in the presence of ligands and PLC drugs. (A) PIE-FCCS data for EGFR-mCH and EphA2-eGFP coexpressed in COS-7 cells. Data were recorded before treatment (white), after the addition of EGF (gray) or EA1 (orange), and after treatment with the PLC inhibitor U73122 (blue) or with the PLC activator 3M3FBS (green). (B) Diffusion coefficients of EGFR-mCH after treatment with ligands and PLC-regulating drugs. (C) Diffusion coefficients of EphA2-eGFP after treatment with ligands and PLC-regulating drugs. (D) Representative epifluorescence images from each set of experiments. One-way ANOVA tests with uncorrected Fisher’s LSD post hoc tests show the individual P values (*P < 0.0259, **P < 0.003, and ****P < 0.0001). Diffusion coefficient data are represented as mean values ± SEM.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Diffusion-based Assay

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 4. EGFR and EphA2 phosphorylation changes after treatment with ligand and PLC drugs. (A) Representative Western blots after treatment with EA1 (15 min, 500 ng/ml), 3M3FBS (45 min, 25 μM), or 3M3FBS followed by EA1. (B) Representative Western blots after treatment with U73122 (15 min, 5 μM), EA1 (as above), EGF (15 min, 100 ng/ml), or U73122 followed by each ligand. (C) Quantification of EphA2 pS897 levels in all conditions normalized to the corresponding total EphA2 bands. N = 6 to 12. Statistical analysis was performed using a Kruskal-Wallis test [H(7) = 19.45, P = 0.007] with a Mann-Whitney U test for comparisons between groups. Signifi- cance values were adjusted by the Bonferroni correction for multiple tests. (D) Quantification of total EphA2 levels in all conditions normalized to actin. N = 6 to 12. Sta- tistical analysis was performed using a Kruskal-Wallis test [H(7) = 38.44, P = 3.0 × 10−6] with a Mann-Whitney U test for comparisons between groups. Significance values were adjusted by the Bonferroni correction for multiple tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 5. AlFoM model of the heterodimer of EGFR residues 669 to 1210 (cyan) and EphA2 residues 559 to 976 (purple). Predictions are shown for EphA2 WT (A) or for the S897E/S901E mutant (B) representative of phosphorylation at serine residues (red bubbles). Yellow residues indicate the active site of each kinase region. The purple halo encompasses the EphA2 SAM domain, while the cyan halo shows the EGFR JM domain. The red arrow indicates the EphA2 kinase active site, which is steri- cally blocked by the EGFR kinase domain in (A), but not in (B).

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Mutagenesis, Phospho-proteomics

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 6. Comparison of EGFR and EphA2 interactions with the lipid bilayer. (A) Snapshots illustrating the predominant conformation of the EGFR-EphA2 heterodimer after molecular dynamics simulations. The intracellular view highlights the EGFR-EphA2 heterodimer’s association with the lipid bilayer, where EGFR is shown in salmon and EphA2 in purple-blue surfaces. Cholesterol, PIP2, and POPS are depicted as cyan, green, and yellow spheres, respectively, while POPC is displayed as gray lines for clarity. The image on the right shows the interaction interface surface of the heterodimer viewed from the intracellular side. (B) Comparison of the number of contacts between EGFR and EphA2 with PIP2 in the membrane over the course of the simulation. The number of contacts is averaged across four replica simulations, considering a 6-Å cutoff distance. KD, kinase domain; SAM, sterile alpha motif.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Comparison, Membrane, Sterility

Journal: Science advances

Article Title: Phosphatidylinositol 4,5-bisphosphate drives the formation of EGFR and EphA2 complexes.

doi: 10.1126/sciadv.adl0649

Figure Lengend Snippet: Fig. 7. Model for EGFR-EphA2 heterodimerization. When PIP2 levels are normal, EGFR (blue) and EphA2 (orange) do not interact in the absence of ligand. When EGF is present, active heterodimer forms in which EGFR Y1068 and EphA2 S897 are phosphorylated. The active conformation of the ICDs allows a more open EphA2 kinase domain with the EGFR JM free to move away from the membrane. Under increased PIP2 conditions, PIP2 promotes inactive heterodimer formation. The inac- tive dimer is likely stabilized by the EGFR JM electrostatically interacting with the membrane, and the EGFR ICD is pulled toward the membrane to close the EphA2 kinase domain.

Article Snippet: Human EphA2 cDNA (residues 1 to 971 based on NM_004431.4) with C- terminal GFP and mCH tag was purchased from Origene in gateway donor vectors as described previously (61).

Techniques: Membrane

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Sample Prep, Membrane, Microscopy, Imaging

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: Western blot analysis of EphA2 pS897 in HEK293T (A) , A375 (B) and A431 (C) cells. We show pS897 quantification (mean ± S.D) and representative blots. p -values from one-way ANOVA followed by Mann-Whitney U or t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) cAMP quantification in HEK293T cells transduced with Red Up cADDis cAMP biosensor ollowing treatment with MβCD. (B) Western blot analysis and quantification of PKA T197 phosphorylation in A375 cells following treatment with MβCD. (C-D) Western blot analysis and quantification of EphA2 S897 phosphorylation in A375 cells following treatment with forskolin and soproterenol, respectively. Data shown in panel B are normalized to the respective total PKA signal in Figure S15A. Data shown in panels C-D are normalized to the respective total EphA2 signal in Figure S15B-C. Quantitative comparisons between treatments were made with respect to normalized control conditions. Bar graphs show mean ± S.D., p -values in panels A-D are from an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

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

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) In the presence of normal levels of Chol (yellow oval), EphA2 (blue) can be found as a monomer and displays low Ser897 phosphorylation. We propose that in this state the kinase domain interacts with the SAM domain. (B) When Chol content is reduced, β-AR (purple) activity increases, promoting cAMP/PKA signaling that enhances Ser897 phosphorylation (red dot). This forces the kinase-SAM linker into an open conformation, which promotes higher-order oligomers independent from ligand stimulation.

Article Snippet: Plasmid encoding full-length EphA2 containing a C-terminal turboGFP tag was obtained from Origene (Accession number: RG205725 ).

Techniques: Phospho-proteomics, Activity Assay

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques: Amplification

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques: Control, Immunohistochemistry, Expressing, Staining

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques:

Journal: Oncogenesis

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

doi: 10.1038/s41389-019-0159-6

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

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

Techniques: Binding Assay

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Representative western blot showing EphA2 phosphorylation in Caveolin-1 (CAV1) silenced clones of A673, RDES, and A4573 cells. SCR = control scrambled sequence. (B) Representative western blot showing total EphA2 expression and its phosphorylation at S897 residue in a panel of ES cell lines. (C) WST1 tetrazolium-based proliferation assay comparing cell lines with high levels of p-EphA2S897 (A4573, A673, and TC252) versus those with low p-EphA2S897 (EW7, TC71, and SK-N-MC). (D) Migration assay in Boyden chambers in the selected panel of high or low p-EphA2S897 expression cell lines. A4573 cell line was set as reference. (E) Sample micrographs from the Ewing sarcoma tissue microarray showing differential expression pattern of p-EphA2S897. Magnification: 40×. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments: *p ≤ 0.05 **p ≤ 0.01. (F) Kaplan-Meier curve comparing differential survival of Ewing sarcoma patients in function of p-EphA2S897 expression. Long-rank (Mantel-Cox test) analysis was used to generate p value.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Western Blot, Clone Assay, Sequencing, Expressing, Proliferation Assay, Migration, Microarray

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Western blot showing EphA2 expression and its phosphorylation at S897 in RH1 reintroduction model. CMV = empty vector. (B) WST1 tetrazolium-based proliferation assay for the RH1 EphA2 reintroduction model. (C) Migration assay in Boyden chambers for the RH1 EphA2 reintroduction model. REphWT was set as reference. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments: *p ≤ 0.05 **p ≤ 0.01.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Western Blot, Expressing, Plasmid Preparation, Proliferation Assay, Migration

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Representative western blot showing EphA2 expression in silencing models generated from A673 and TC252 cell lines. (B) Proliferation assay using the WST1 tetrazolium-based assay. (C) Quantification of clonogenic assay. Representative images are shown above the bars. “Clone A” and “B” denote clones AshE3 and TshE26 and AshE15 and TshE35, respectively. (D) In vivo tumor growth in immunodepressed mice after subcutaneous injection of cells. Tumor size at the time of euthanasia is shown; n=8. SCR = control scrambled sequence. AshE#/TshE# = EphA2 silenced selected clones. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments: *p ≤ 0.05 **p ≤ 0.01.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Western Blot, Expressing, Generated, Proliferation Assay, Clonogenic Assay, Clone Assay, In Vivo, Injection, Sequencing

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Migration assay in Boyden chambers using EphA2 silenced models. “Clone A” and “B” denote clones AshE3 and TshE26 and AshE15 and TshE35, respectively. SCR models were set as reference. (B) Invasion assay in Matrigel-coated Boyden chambers using EphA2 silenced models. “Clone A” and “B” denote clones AshE3 and TshE26 and AshE15 and TshE35, respectively. SCR models were set as reference. (C) Lung metastasis incidence in immunodepressed mice after tail-vein injection of control or EphA2 silenced A673 cells; n = 10. (D) Lung metastasis incidence in immunodepressed mice after injection of tumor cells in the gastrocnemius; n = 7. SCR = control scrambled sequence. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments. Fisher’s exact test was used for evaluating differences in lung metastasis incidence in mice: *p ≤ 0.05 **p ≤ 0.01.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Migration, Clone Assay, Invasion Assay, Injection, Sequencing

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Representative western blot showing ERK, p-ERK, Akt, and p-AktS473 levels in EphA2 silenced clones of the A673 cell line. SCR = control scrambled sequence. AshE#/TshE# = EphA2 silenced selected clones. (B–C) Representative western blots showing EphA2 and p-EphA2S897 levels after U0126 (B) and Trametinib (C) treatments in A673 and TC252 cell lines. Phosphorylation of ERK (B and C) is shown as a control of treatment efficiency. (D) Representative western blot showing EphA2, p-EphA2897, ERK, and p-ERK in the RH1 reintroduction model. CMV = empty vector. (E) Migration assay in Boyden chambers for A673 and TC252 cell lines after 50 nM Trametinib treatment. DMSO (vehicle) conditions were set as reference. (F) Invasion assay in Matrigel-coated Boyden chambers for A673 and TC252 cell lines after 50 nM Trametinib treatment. DMSO (vehicle) conditions were set as reference. (G) Migration assay in Boyden chambers in the RH1 EphA2 reintroduction model. REphWT plus DMSO (vehicle) condition was set as reference. (H) Invasion assay in Matrigel-coated Boyden chambers in the RH1 EphA2 reintroduction model. REphWT plus DMSO (vehicle) condition was set as reference. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments. Fisher’s exact test was used for evaluating differences in lung metastasis incidence in mice: *p ≤ 0.05 **p ≤ 0.01 ***p ≤ 0.001.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Western Blot, Clone Assay, Sequencing, Plasmid Preparation, Migration, Invasion Assay

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A) Heatmap of up- (red) and down- (blue) regulated genes in control cells vs. EphA2 silenced cells. (B) Venn Diagram showing the number of common genes differentially expressed after EphA2 knockdown in A673 and TC252 cell lines. (C) Gene Set Enrichment Analysis (GSEA) showing a correlation between genes differentially expressed after EphA2 silencing and those involved in metastasis. NES = Normalized Enrichment Score. (D) Ingenuity pathway analysis showing cellular processes significantly altered by EphA2 knockdown.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques:

Journal: International journal of cancer

Article Title: EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma

doi: 10.1002/ijc.31405

Figure Lengend Snippet: (A–B) Validation of up- (A) and down- (B) regulated targets after EphA2 silencing by RT-qPCR in A673 and TC252 models. CTRL represents the mean from both the parental and the control vector transfected pool. shEphA2 represents the mean from the two representative clones employed through the previous experiments. (C) Migration assay in Boyden chambers after ADAM19 silencing in A673 cell line. siCTRL (= non-targeting siRNA) was set as reference. (D) Representative western blot showing ADAM19 expression and phosphorylation of EphA2 and ERK after siRNA silencing in A673 cell line. siCTRL = non-targeting siRNA. (E) Representative western blot showing ADAM19 expression and EphA2 phosphorylation after treatment with 50 nM Trametinib in the A673 cell line. ERK and p-ERK are shown as treatment efficiency controls. (F) Migration assay in Boyden chambers after ADAM19 silencing in RH1 EphA2 reintroduction model. REphWT plus siCTRL (= non-targeting siRNA) was set as reference. Data are presented as means ± SD. Statistical significance was achieved by the Student’s t test from at least three different experiments: *p ≤ 0.05 **p ≤ 0.01.

Article Snippet: RH1 transfected cells stably expressing pCMV6-EphA2 Myc-DKK tagged vector (Origene #RC205725) were selected with 0.6 mg/mL of neomycin (Life Technologies).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Transfection, Clone Assay, Migration, Western Blot, Expressing