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: 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