Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: Proliferative effects of VEGFR2 R1032Q expression in VEGFR2 negative vs VEGFR2 expressing cell models. a , Western blot (WB) analysis of total VEGFR2 levels in MCF7 or Sk-Mel-31 cells. Total lysates of non-transfected (NT) cells and of cells transfected to express VEGFR2 WT (WT) or VEGFR2 R1032Q (R1032Q) were analyzed. FAK and α-tubulin levels were measured as loading controls. Images are representative of three independent experiments that gave superimposable results. b , 2D MCF7 cell proliferation. Mean cell number of three biological replicates. c , 2D Sk-Mel-31 cell proliferation. Mean covered area ± SEM of N = 24 biological replicates from three independent experiments. *, p < 0.05, ****, p < 0.001, One-Way ANOVA followed by Dunnett’s post-hoc test. d , 3D anchorage-independent MCF7 cell proliferation. Average size of N = 300-1200 cell aggregates from two independent experiments. Representative images, scale bar 200 μm. e , 3D anchorage-independent Sk-Mel-31 cell proliferation. Average size of N = 114-142 cell colonies from two independent experiments. Representative microphotographs, scale bar 200 μm. f-g , in vivo growth of Sk-Mel-31-VEGFR2 WT and Sk-Mel-31-VEGFR2 R1032Q cells injected subcutaneously into the flank of NOD/SCID mice ( N = 11-12). Tumor volume after 35 days ( f ). Immunofluorescence analysis of pVEGFR2 (in green) in tumor FFPE sections at day 35 after implantation. TO-PRO-3 nuclei counterstaining is shown in magenta. Scale bar, 20 μm ( g ). *, p < 0.05, **, p < 0.01,***, p < 0.005, ****, p < 0.001, Student’s t-test.

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Expressing, Western Blot, Transfection, In Vivo, Injection, Immunofluorescence

Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: VEGFR2 R1032Q forms functional heterodimers with co-expressed VEGFR2 WT . a , WB analysis of pVEGFR2 (Y1175) and total VEGFR2 levels in total lysates of serum-starved CHO cells (NT) or expressing VEGFR2 WT (WT) or VEGFR2 R1032Q (R1032Q) in the absence or in the presence of 30 ng/mL VEGF. FAK levels were measured as loading control. b , WB analysis using anti-VEGFR2 of total p-Tyr- (up) and Anti-VEGFR2 immunocomplexes (IP) (down) of serum-starved CHO cells expressing VEGFR2 WT or VEGFR2 R1032Q or the kinase-dead mutant VEGFR2 K868M (K868M) using antibodies anti VEGFR2. WB images are representative of three independent experiments that gave superimposable results. c , WB analysis of phospho-VEGFR2 (Tyr1175), VEGFR2 and YFP in total lysates of serum-starved CHO cells expressing YFP-tagged VEGFR2 WT alone or in combination with untagged VEGFR2 WT or VEGFR2 R1032Q or untagged VEGFR2 R1032Q alone. Yellow arrowheads, YFP-tagged VEGFR2. Black arrowheads, untagged VEGFR2. The densitometric quantification of 3 independent experiments is shown in the bar graph. d , FRET efficiency of mCherry-VEGFR2 WT -YFP-VEGFR2 WT or mCherry-VEGFR2 WT -YFP-VEGFR2 R1032Q couples expressed in CHO cells measured by FLIM. Representative color-coded FRET efficiency images of cell membrane ROI. FRET efficiency of the mCherry-VEGFR2 WT -YFP-VEGFR2 WT couple treated with VEGF was used as control. e , measurement of the kinase activity of PtA-adsorbed VEGFR2-immunocomplexes isolated from CHO cells expressing VEGFR2 WT or VEGFR2 R1032Q before (Ctrl) or after the incubation with the total lysate of CHO cells expressing VEGFR2 WT or VEGFR2 R1032Q to induce receptor dimerization. f , phosphorylation levels of PtA-adsorbed GFP-immunocomplexes isolated from CHO cells expressing VEGFR2 WT(YFP) or VEGFR2 R1032Q(YFP) before (Ctrl) or after the incubation with the total lysate of CHO cells expressing VEGFR2 WT or VEGFR2 R1032Q to induce receptor dimerization. *, p < 0.05; **, p < 0.01; ***, p < 0.005, One-Way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Functional Assay, Expressing, Control, Mutagenesis, Membrane, Activity Assay, Isolation, Incubation, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: Co-expression of VEGFR2 WT -VEGFR2 R1032Q alters the recruitment of intracellular signaling molecules. Total lysates of CHO cells transiently transfected with VEGFR2 WT or with VEGFR2 WT + VEGFR2 R1032Q were used to immunoprecipitate VEGFR2. a ,immunoprecipitated fractions were analyzed by WB for the presence of Shp2, Nck and Grb2. Non-adjacent lanes of the same blot. b , WB densitometry and statistical analysis of 2 independent experiments was performed. *, p < 0.05; **, p < 0.01; Student’s t Test vs VEGFR2 WT .

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Expressing, Transfection, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: The heterodimeric VEGFR2 WT -VEGFR2 R1032Q complex displays altered lateral membrane mobility. Diffusion rate ( a ) and immobile fraction ( b ) of YFP-tagged VEGFR2 variants (WT YFP or R1032Q YFP ) alone or upon co-expression with untagged VEGFR2 variants as measured by FRAP analyses on the cell surface of transfected CHO cells. Stimulation with 30 ng/mL of VEGF of CHO cells expressing YFP-VEGFR2 WT was used as control. Data are shown as mean ± SEM of 2 independent experiments. Individual values of each replicate are shown. *, p < 0.05, ***, p < 0.005, One-Way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Membrane, Diffusion-based Assay, Expressing, Transfection, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: VEGFR2 distribution within membrane microdomains. Western blot analysis of the levels of flotillin 1 (Flot1), early endosome antigen 1 (EEA1), total VEGFR2, and phospho-VEGFR2 (Tyr 1175) in DRM vs DSM membrane fractions of CHO cells expressing VEGFR2 WT , VEGFR2 R1032Q or the couple VEGFR WT -VEGFR2 R1032Q . CHO- VEGFR2 WT was treated with 30 ng/mL of VEGF.

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Membrane, Western Blot, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Cancer-associated VEGFR2 R1032Q sustains receptor activation also by promoting ligand-independent hetero-dimerization with co-expressed wild-type VEGFR2 and translocation into lipid rafts

doi: 10.1016/j.neo.2025.101195

Figure Lengend Snippet: Effect of VEGFR2 WT -VEGFR2 R1032Q co-expression on the lipid profile of CHO cells. a , analysis of total lipid content (SFAs, MUFAs and PUFAs) of CHO cells expressing VEGFR2 WT , VEGFR2 R1032Q or the couple VEGFR WT -VEGFR2 R1032Q treated with 30 ng/mL of VEGF or left untreated. b , analysis of SFA, SM and cholesterol in DRM vs DSM fractions of CHO cells expressing VEGFR2 WT , VEGFR2 R1032Q or the couple VEGFR WT -VEGFR2 R1032Q treated with 30 ng/mL of VEGF or left untreated. *, p < 0.05; **, p < 0.01, One-Way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: To evaluate whether VEGFR2 R1032Q can acquire enzymatic activity upon dimerization with VEGFR2 WT , the kinase activity of proteinA Sepharose (PtA)-adsorbed VEGFR2 WT or VEGFR2 R1032Q immunocomplexes was measured before and after incubation with total lysates of VEGFR2 WT - or VEGFR2 R1032Q -expressing CHO cells.

Techniques: Expressing