Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: EphA2 antibody, anti-mouse, APC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-109-187 RRID: AB_2651638.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context *

doi: 10.1074/mcp.M113.030528

Figure Lengend Snippet: Signaling dynamics from time resolved tyrosine phosphorylation measurements by quantitative mass spectrometry. Of the 133 manually curated phosphorylation sites, only 37 had a full time course data set, with 22 phosphosites having at least two readings per time point in both treatment conditions. The phosphorylation level of these sites is provided for two different treatment conditions: 20 ng/ml VEGF (VEGF), and 20 ng/ml VEGF + 500 ng/ml PF-4 (VEGF + PF-4). Raw signal measurements were normalized to total protein values and reported as relative proportions to a master lysate stimulated with saturating 50 ng/ml VEGF. Notable temporal increases in signaling dynamics following PF-4 cotreatment were observed for Annexin A2(Y24), EphA2(Y772), Fyn(Y420), GSK3β (Y216), Lyn(Y397), P38α(Y182), Paxillin(Y118), and SHC1(Y318). Attenuations in signaling were noticed for Annexin I(Y21), DYRK1B(Y273), and focal adhesion kinase FAKY576) with PF-4 present. Changes were not mutually exclusive in the same time course data as both increases and decreases in phosphoprotein levels were observed at different time points for many of these proteins as well as Cdk1(T14/Y15) and MPZL1(Y263). Treatment with PF-4 alone yielded noninformative results. Statistical analyses performed using Student's t test found a majority of statistically significant differences to occur in early time points (first 60 min), indicating that early signaling most likely informs endothelial response. Errors bars represent standard error. Statistical significance indicated by asterisks: * for p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Membranes were blocked with 5% BSA in 0.1% Tween in PBS (PBS-T) and incubated with antibodies for β-actin (1:5000), α/β tubulin (1:5000), phospho-ERK1/2 (1:5000), phosphoY772-EphA2 (Tyr772) (1:1000) (Cell Signaling Technology, Beverly, MA), and EphA2 (1:1000) (Cell Applications, San Diego, CA) overnight at 4 °C.

Techniques: Phospho-proteomics, Mass Spectrometry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context *

doi: 10.1074/mcp.M113.030528

Figure Lengend Snippet: Important phosphorylation sites involved with PF-4 and VEGF crosstalk and their previously reported functions. A subset of the phosphorylation sites found to be involved with PF-4 and VEGF signaling in the sprouting angiogenesis data set is highlighted in this table, including 22 of the 37 phosphoproteins that were found to have complete coverage across all time points in the data set. Previously reported functions for these proteins and phosphorylations are shown from different cell systems, as well as some that have been reported to play roles in angiogenesis. Although they may have been reported to have roles in other cell phenotypes, there may be overlap with the PF-4 and VEGF signaling system, such as the potential importance of cell migration and polarization in orchestrating blood vessel formation

Article Snippet: Membranes were blocked with 5% BSA in 0.1% Tween in PBS (PBS-T) and incubated with antibodies for β-actin (1:5000), α/β tubulin (1:5000), phospho-ERK1/2 (1:5000), phosphoY772-EphA2 (Tyr772) (1:1000) (Cell Signaling Technology, Beverly, MA), and EphA2 (1:1000) (Cell Applications, San Diego, CA) overnight at 4 °C.

Techniques: Phospho-proteomics, Migration, Binding Assay, Activation Assay, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context *

doi: 10.1074/mcp.M113.030528

Figure Lengend Snippet: Correlative network connections between significant phosphosites involved with the VEGF and PF-4 signaling. Significant pairwise correlations from above ( p < 0.05 ) were compared between the two treatment conditions where HDMVECs were dosed by VEGF alone or in conjunction with PF-4. To identify potential dynamic changes in the topography of signaling, the presence or absence of correlations between the two conditions were mapped out. Black edges represent strong positive correlations present in both the presence and absence of PF-4 co-treatments with VEGF. Red edges indicate correlations present when endothelial cells were dosed with VEGF only. Green edges indicate correlations that appeared only when HDMVECs were simultaneously treated with PF-4 and VEGF and not with VEGF only. There were a larger number of correlations that appeared only with VEGF treatment, such as those centered around CD31 via shear stress response as well as migratory-centric pathways involving FAK and Src Family Kinases. As expected by many of the reported angiostatic effects of PF-4, red edge correlations (VEGF only) disappeared for FAK and other migratory pathways. Interestingly, many new green edge correlations (VEGF + PF-4) appeared around EphA2, contrary to its previously reported roles in promoting angiogenesis.

Article Snippet: Membranes were blocked with 5% BSA in 0.1% Tween in PBS (PBS-T) and incubated with antibodies for β-actin (1:5000), α/β tubulin (1:5000), phospho-ERK1/2 (1:5000), phosphoY772-EphA2 (Tyr772) (1:1000) (Cell Signaling Technology, Beverly, MA), and EphA2 (1:1000) (Cell Applications, San Diego, CA) overnight at 4 °C.

Techniques: Shear

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context *

doi: 10.1074/mcp.M113.030528

Figure Lengend Snippet: Western blots of phosphorylated and total EphA2 indicate increases in both following co-treatment of HDMVECs with PF-4 at 30 min. Representative Westerns ( A ) are shown for both phospho- and total EphA2. Semiquantitative analysis of Western blots using pixel intensity data ( n = 7) yielded increases in both total and phospho-EphA2 when PF-4 was added ( B ). The phospho-EphA2 increase agrees with the signaling data obtained using mass spectrometry. These data indicate the potential role that EphA2 may play in mediating the signaling crosstalk effects by EphA2. Although there was no statistical significance found in phosphorylation levels between VEGF and VEGF with PF-4 treatment, statistical significance was found for total EphA2 levels between the two treatment conditions ( p < 0.05 , Wilcoxon rank sum test). Error bars represent standard error.

Article Snippet: Membranes were blocked with 5% BSA in 0.1% Tween in PBS (PBS-T) and incubated with antibodies for β-actin (1:5000), α/β tubulin (1:5000), phospho-ERK1/2 (1:5000), phosphoY772-EphA2 (Tyr772) (1:1000) (Cell Signaling Technology, Beverly, MA), and EphA2 (1:1000) (Cell Applications, San Diego, CA) overnight at 4 °C.

Techniques: Western Blot, Mass Spectrometry, Phospho-proteomics

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Liposomal Nanostructures for Drug Delivery in Gastrointestinal Cancers

doi: 10.1124/jpet.118.254797

Figure Lengend Snippet: Selected liposomal products in clinical development in GI cancers

Article Snippet: Solid tumors including gastric/gastroesophageal/pancreatic ductal adenocarcinoma , (MM-310) docetaxel in liposomes targeted with antibodies to EphA2 receptor , I , Merrimack Pharmaceuticals , {"type":"clinical-trial","attrs":{"text":"NCT03076372","term_id":"NCT03076372"}} NCT03076372.

Techniques: Plasmid Preparation