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: Journal of cell science

Article Title: HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

doi: 10.1242/jcs.163790

Figure Lengend Snippet: Fig. 1. Knockdown of EphA2 suppresses HGF- induced formation of extensions in MDCK cysts. (A) MDCK cells were grown in Matrigel for 10 days, then they were fixed and stained with an antibody against EphA2 antibody (green) and phalloidin (red, labeling for F-actin to visualize cyst structure). (B) Cell lysates from MDCK cells expressing a control shRNA against luciferase (shControl) or an shRNA against EphA2 (shEphA2) were analyzed by immunoblotting with antibodies against EphA2 and α-tubulin. (C) MDCK cells were grown in Matrigel for 10 days, and the cyst lysates were analyzed by immunoblotting with antibodies against EphA2 and α-tubulin. (D) MDCK cells expressing shControl or shEphA2 were grown in Matrigel for 10 days and then fixed. Cysts were stained with an antibody against EphA2 (green) and phalloidin (red). (E) MDCK cysts were stained with phalloidin. (F) Quantification of the percentage of normal cysts. Data are the means±s.e.m. from three independent experiments (ns, not significant; one-way ANOVA with Dunnett T3). (G) MDCK cells expressing shControl or shEphA2 were grown in Matrigel for 8 days, followed by treatment with 50 ng/ml HGF for 48 hours. Cysts were stained with phalloidin. Low mag., low magnification. (H) Quantification of cysts with one or more actin-rich extensions. Data are the means±s.e.m. of three independent experiments (*P<0.05; ns, not significant; one-way ANOVA with Dunnett T3). At least 100 cysts were counted for each experiment. Scale bars: 50 µm.

Article Snippet: The following antibodies were used in this study: a mouse monoclonal antibody against Myc, a rabbit polyclonal antibody against EphA2 (C-20), and a rat monoclonal antibody against RhoG (1F3 B3 E5) (Santa Cruz Biotechnology); mouse monoclonal antibodies against Flag (M2) and αtubulin (Sigma); a rabbit polyclonal antibody against phosphorylated EphA2 (at residue S897) (Cell Applications); rabbit monoclonal antibodies against phosphorylated EphA2 (at residue S897; clone D9A1) and phosphorylated EphA2 (at residue Y588; clone D7X2L, Cell Signaling); a mouse polyclonal antibody against EphA2 (D7, Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); and secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen).

Techniques: Knockdown, Staining, Labeling, Expressing, Control, shRNA, Luciferase, Western Blot

Journal: Journal of cell science

Article Title: HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

doi: 10.1242/jcs.163790

Figure Lengend Snippet: Fig. 2. Phosphorylation of EphA2 on S897 is required for HGF-induced formation of extensions in MDCK cysts. (A) The EphA2 constructs used in this study. TM, transmembrane; KD, kinase domain; SAM, sterile-α-motif. Numbers indicate amino acid position within the sequence. (B) Lysates from MDCK cells expressing the indicated shRNAs or co-expressing the EphA2 constructs (-WT, -SA or -KM) were analyzed by immunoblotting with antibodies against EphA2 and α-tubulin. (C) MDCK cells were grown in Matrigel for 10 days, and the cyst lysates were analyzed by immunoblotting with antibodies against EphA2, EphA2 phosphorylated at S897 (pS897-EphA2) or at Y588 (pY588-EphA2) and α-tubulin. (D) MDCK cysts expressing the indicated plasmids were treated with HGF for 48 hours and then fixed. Cysts were stained with phalloidin. Low mag., low magnification. (E) Quantification of cysts with one or more actin-rich extensions. Data are the means±s.e.m. of four independent experiments (*P<0.05; **P<0.01; ns, not significant; one-way ANOVA with Dunnett T3), and at least 100 cysts were counted for each experiment. Scale bars: 50 µm.

Article Snippet: The following antibodies were used in this study: a mouse monoclonal antibody against Myc, a rabbit polyclonal antibody against EphA2 (C-20), and a rat monoclonal antibody against RhoG (1F3 B3 E5) (Santa Cruz Biotechnology); mouse monoclonal antibodies against Flag (M2) and αtubulin (Sigma); a rabbit polyclonal antibody against phosphorylated EphA2 (at residue S897) (Cell Applications); rabbit monoclonal antibodies against phosphorylated EphA2 (at residue S897; clone D9A1) and phosphorylated EphA2 (at residue Y588; clone D7X2L, Cell Signaling); a mouse polyclonal antibody against EphA2 (D7, Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); and secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen).

Techniques: Phospho-proteomics, Construct, Sterility, Sequencing, Expressing, Western Blot, Staining

Journal: Journal of cell science

Article Title: HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

doi: 10.1242/jcs.163790

Figure Lengend Snippet: Fig. 3. EphA2 is phosphorylated on S897 through HGF in MDCK cysts. (A) MDCK cells were grown in Matrigel for 8 days and then treated with HGF for 48 hours in the presence or the absence of 20 µM LY294002 or 1 µM MK- 2206. The cyst lysates were analyzed by immunoblotting with antibodies against EphA2, pS897-EphA2 and α-tubulin. The relative amount of pS897-EphA2 was determined by the amount of pS897-EphA2 normalized to the amount of total EphA2 in cyst lysates, analyzed by using ImageJ software. Data are presented as the means±s.e.m. from four independent experiments (*P<0.05; **P<0.01; one-way ANOVA with Dunnett T3). (B) MDCK cells were grown in Matrigel for 8 days, followed by treatment with HGF for 48 hour in the presence and the absence of 20 µM LY294002. Low mag., low magnification. (C) Quantification of cysts with one or more actin-rich extensions. Data are the means±s.e.m. of four independent experiments (***P<0.001; Student’s t-test), and at least 100 cysts were counted for each experiment. (D) Following treatment with HGF for 48 hours, MDCK cysts were stained with phalloidin (red) and antibodies against pS897-EphA2 or EphA2 (green). Scale bars: 50 µm.

Article Snippet: The following antibodies were used in this study: a mouse monoclonal antibody against Myc, a rabbit polyclonal antibody against EphA2 (C-20), and a rat monoclonal antibody against RhoG (1F3 B3 E5) (Santa Cruz Biotechnology); mouse monoclonal antibodies against Flag (M2) and αtubulin (Sigma); a rabbit polyclonal antibody against phosphorylated EphA2 (at residue S897) (Cell Applications); rabbit monoclonal antibodies against phosphorylated EphA2 (at residue S897; clone D9A1) and phosphorylated EphA2 (at residue Y588; clone D7X2L, Cell Signaling); a mouse polyclonal antibody against EphA2 (D7, Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); and secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen).

Techniques: Western Blot, Software, Staining

Journal: Journal of cell science

Article Title: HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

doi: 10.1242/jcs.163790

Figure Lengend Snippet: Fig. 4. EphrinA1 dephosphorylates EphA2 on S897 and suppresses extension formation in MDCK cysts. (A) MDCK cells grown in Matrigel were treated with 1 µg/ml ephrinA1 for 24 hours in the presence of HGF and then stained with phalloidin. Low mag., low magnification. Scale bars: 50 µm. (B) Quantification of cysts with one or more actin-rich extensions. Data are the means±s.e.m. of three independent experiments (***P<0.001; Student’s t-test), and at least 100 cysts in randomly selected fields were counted in one experiment. (C) MDCK cysts were treated with 1 µg/ml ephrinA1 for 24 hours in the presence of HGF. The cyst lysates were analyzed by immunoblotting with antibodies against EphA2, EphA2 phosphorylated at S897 (pS897- EphA2) or at Y588 (pY588-EphA2) and α-tubulin. The relative levels of pS897-EphA2 and pY588-EphA2 were determined by the amount of pS897-EphA2 or pY588-EphA2 normalized to the amount of total EphA2 in cyst lysates, as analyzed by using ImageJ software. Data are presented as the means±s.e.m. from four independent experiments (*P<0.05; ns, not significant; one-way ANOVA with Dunnett T3). (D) MDCK cells expressing the indicated plasmids were grown in Matrigel for 8 days and then treated with HGF for 48 hours. The cyst lysates were analyzed by immunoblotting with antibodies against EphA2, pY588-EphA2 and α-tubulin.

Article Snippet: The following antibodies were used in this study: a mouse monoclonal antibody against Myc, a rabbit polyclonal antibody against EphA2 (C-20), and a rat monoclonal antibody against RhoG (1F3 B3 E5) (Santa Cruz Biotechnology); mouse monoclonal antibodies against Flag (M2) and αtubulin (Sigma); a rabbit polyclonal antibody against phosphorylated EphA2 (at residue S897) (Cell Applications); rabbit monoclonal antibodies against phosphorylated EphA2 (at residue S897; clone D9A1) and phosphorylated EphA2 (at residue Y588; clone D7X2L, Cell Signaling); a mouse polyclonal antibody against EphA2 (D7, Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); and secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen).

Techniques: Staining, Western Blot, Software, Expressing

Journal: Journal of cell science

Article Title: HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture.

doi: 10.1242/jcs.163790

Figure Lengend Snippet: Fig. 6. EphA2 mediates HGF-induced RhoG activation. (A,B) MDCK cells expressing the indicated plasmids were treated with 50 ng/ml HGF for 1 minute at room temperature. Cell lysates were incubated with GST-ELMO-NT, and bound endogenous RhoG and total cell lysates were analyzed using antibodies against RhoG and EphA2. Relative RhoG activity was determined by the amount of RhoG bound to GST-ELMO-NT normalized to the amount of RhoG in cell lysates, as analyzed by using ImageJ software. Data are presented as the means±s.e.m. from three or four independent experiments (*P<0.05; one-way ANOVA with Dunnett T3). (C) MDCK cells expressing shEphA2 or co-expressing GFP-tagged RhoG- V12 were treated with HGF for 48 hours and stained using phalloidin. Low mag., low magnification. (D) Quantification of cysts with one or more actin-rich extensions. Data were the means±s.e.m. of three independent experiments (*P<0.05; ns, not significant; one-way ANOVA with Dunnett T3), and at least 100 cysts were counted for each experiment. Scale bars: 50 µm.

Article Snippet: The following antibodies were used in this study: a mouse monoclonal antibody against Myc, a rabbit polyclonal antibody against EphA2 (C-20), and a rat monoclonal antibody against RhoG (1F3 B3 E5) (Santa Cruz Biotechnology); mouse monoclonal antibodies against Flag (M2) and αtubulin (Sigma); a rabbit polyclonal antibody against phosphorylated EphA2 (at residue S897) (Cell Applications); rabbit monoclonal antibodies against phosphorylated EphA2 (at residue S897; clone D9A1) and phosphorylated EphA2 (at residue Y588; clone D7X2L, Cell Signaling); a mouse polyclonal antibody against EphA2 (D7, Millipore); secondary antibodies conjugated to horseradish peroxidase (DAKO); and secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen).

Techniques: Activation Assay, Expressing, Incubation, Activity Assay, Software, Staining