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: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: BioID protein interaction profiling reveals a PI3Kβ–EPHA2 interaction induced by PTEN loss in cancer cells. A, Workflow depicting the BioID experiment. PTEN-null cancer cells were overexpressed with vector, BioID- PIK3CA , or BioID- PIK3CB , and biotin was added to the culture medium, followed by immunoprecipitating with SBP beads. Biotinylation and identification of bait-interacting protein was detected by MS. B, Heatmap showing the MS intensities of all proteins pulled down by PIK3CA -BirA* or PIK3CB -BirA* in PTEN-null cell lines (MDA-MB-468 and BT549). Clustering was assessed with the Euclidean distance measurement in column and the Z-normalization in row. Proteins of interest were colored. C, Plot of sum intensity for protein interactors of PI3Kα and PI3Kβ identified by MS analysis from cells expressing PIK3CA-BirA* or PIK3CB-BirA* in PTEN-null cell lines (MDA-MB-468 and BT549). D, EPHA2 colocalized with PI3Kβ on the cell membrane. IF staining with anti-PI3Kβ (green) and anti-EPHA2 (red) in PTEN-null BT549 cells. DAPI was used for nuclear staining. Scale bars, 50 μm. E, Endogenous EPHA2 IP from PTEN-null BT549 and HCC70 cells analyzed by Western blot with the indicated antibodies. F, PTEN loss enhanced PI3Kβ–EPHA2 interaction. PTEN-WT and PTEN-KO HEK293T cells were transfected with Flag-tagged PI3Kα, PI3Kβ, and EPHA2-HA, and cell lysate was immunoprecipitated with anti-Flag antibody. G, PTEN restoration blunted the dominant interaction between EPHA2 and PI3Kβ. Co-IP assay of exogenous PI3Kα and PI3Kβ with exogenous EPHA2 in vector and PTEN stably overexpressed BT549 cells. Flag-tagged PI3Kα and PI3Kβ were immunoprecipitated with anti-Flag antibody and then analyzed by Western blot with the indicated antibodies. H, PTEN restoration blunted the dominant interaction between endogenous EPHA2 and PI3Kβ in BT549 cells.

Article Snippet: The lysates were then incubated with anti-Flag M2 agarose (Sigma, cat. #M8823, RRID: SCR_024337), anti-HA M2 agarose (Sigma, cat. #SAE019), or anti-EPHA2 (Cell Signaling Technology, cat. #6997, RRID: AB_10827743) antibody together with protein A agarose (MCE, cat. #HY-K0203) for 3 hours at 4°C.

Techniques: Plasmid Preparation, Expressing, Membrane, Staining, Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection