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: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 1 | High ACE2 expression was linked to increased VM and better prognosis in NSCLC. (A) Typical image of ACE2, VE-cadherin, EphA2 protein expression and CD34/PAS double staining in TMA tissues. Case B1 had massive CD34−/PAS+ VM (yellow arrows) lined by ACE2, VE-cadherin and EphA2 high expressing tumor cells. Case G1 had abundant CD34+/PAS−MVs (black arrows) with ACE2, VE-cadherin and EphA2 low expressing tumor cells. (B) Kaplan-Meier analysis of OS in NSCLC patients with ACE2 low or high expression. P = 0.044.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Double Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 5 | VM formation was increased, and vasculature was lessened due to inhibition of RAS in vivo. (A) Growth curve of allograft tumors of A549-ACE2-OE cells, A549-NC cells with or without ACEI treatment, Mean ± SD, n = 3, *p < 0.05. ns, no significance. (B) Weight of resected tumors, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. (C) Continuous sections of allograft tumor tissues stained with PAS, CD34, VE-cadherin, or EphA2 immunohistochemical stain. Black arrow points out a typical MV (CD34+/PAS−); yellow arrows point out typical VM (CD34−/PAS+). (D) Quantification of MV and VM in different groups, Mean ± SD, n = 3, per field, ***p < 0.001, **p < 0.01. (E) Quantification of VE-cadherin and EphA2 mean optical density in three groups, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Inhibition, In Vivo, Staining, Immunohistochemical staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 6 | VE-cadherin and EphA2 expression was upregulated in A549 cells and NSCLC tissues with impaired local RAS status. (A, B) RT-PCR experiment of VE-cadherin and EphA2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (C–E) Western blot analysis and quantification of VE- cadherin and EphA2 expression level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (F, G) Linear regressions of VM number and VE- cadherin (P < 0.0001) or EphA2 (P = 0.0108) score in TMA. (H) Typical tissue images of both groups stained with VE-cadherin, ACE2 or CD34/PAS. Case F13 with ACE2 low status was provided with rambling VM covered by tumor cells which only expressed VE-cadherin in nuclei; case D6 with ACE2 high status had ordered VM lined by tumor cells expressing VE-cadherin in both nuclei and cytomembranes. Red arrow: VE-cadherin membrane expression.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Staining, Membrane