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

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Confocal microscopic images of OKF6/TERT-2 epithelial cells that had been infected with GFP expressing C. albicans (CAI4-GFP) and then stained for EphA2 (red). Results are representative of 3 independent experiments. Arrows indicate the accumulation of EphA2 around the fungal cells. Hollow arrows indicate organisms that were analyzed for fluorescent intensity in ( b ). Negative control images are shown in ( b ) Magnified image of C. albicans cells with plots of fluorescent intensity at the regions indicated by the dotted line. The green lines indicate the fluorescent intensity of GFP expressing C. albicans and the red lines indicate the fluorescent intensity of the EphA2. ( c ) Immunoblot analysis showing the time course of EphA2 phosphorylation in oral epithelial cells that had been infected with yeast-phase C. albicans SC5314 for the indicated times. ( d ) EphA2 phosphorylation after 15-min infection with either C. albicans yeast or pregerminated hyphae. H, hyphae; Y, yeast. ( e ) Effects of C. albicans (SC5314, 529L, efg1/cph1 ), Candida glabrata , and Saccharomyces cerevisiae on EphA2 phosphorylation. Cg, C. glabrata ; Sc, S. cerevisiae . ( f–h ) Time course (in minutes) of EphA2 phosphorylation induced by zymosan ( f ), laminarin ( g ), and heat-killed C. albicans SC5314 (HK Ca ) ( h ). ( i ) Binding of recombinant EphA2 to immobilized ephrin A1, zymosan, laminarin, mannan, and BSA, as determined by ELISA. Box whisker plots show median and range of 3 experiments, each performed in triplicate. Statistical analysis of binding is shown relative to wells coated with BSA. EFNA1, ephrin A1; lam, laminarin; man, mannan; zym, zymosan ( j ) EphA2 (red) and Fc-dectin-1 (green) bind to exposed β-glucan on yeast-phase C. albicans . Results are representative of 3 independent experiments. Densitometric analyses of replicate immunoblots are shown in ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant (two-tailed Student’s t-test assuming unequal variances). Scale bars 5 μm.

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Infection, Expressing, Staining, Negative Control, Western Blot, Phospho-proteomics, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Whisker Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a–b ) Effects of EphA2 depletion with siRNA on the endocytosis of C. albicans by oral epithelial cells ( a ) and extent of C. albicans -induced host cell damage ( b ). ( c ) Effects of inhibition of EphA2 with dasatinib or an EphA2 antagonist and/or inhibition of the epidermal growth factor receptor (EGFR) with gefitinib on the endocytosis of C. albicans by oral epithelial cells. Box whisker plots show median and range of three experiments, each performed in triplicate. ( d ) Effect of EphA2 siRNA on EGFR phosphorylation at Y1068 in oral epithelial cells infected with C. albicans . ( e ) Effect of inhibition of EGFR with gefitinib on the phosphorylation of epithelial cell EphA2 in response to C. albicans infection. ( f ) Immunoblots showing the effects of EphA2 siRNA on the phosphorylation of epithelial cell MEK1/2 and c-FOS in response to C. albicans . ( g ) Effect of treatment of epithelial cells with an anti-dectin-1 mAb on the phosphorylation of p65 S536 induced by C. albicans. ( h ) Time course (in min) of MEK1/2 phosphorylation induced by zymosan. ( i ) Effect of EGFR inhibition on MEK1/2 phosphorylation in response to C. albicans . ( j ) Transient MEK1/2 activation in response to heat-killed C. albicans . Densitometric analyses of replicate immunoblots are shown in ).ANT, EphA2 antagonist; DAS, dasatinib; GEF, gefitinib; p.i., post-infection; HK Ca , Heat-killed C. albicans ; zym, zymosan. **** P < 0.0001 (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Inhibition, Whisker Assay, Phospho-proteomics, Infection, Western Blot, Activation Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Immunoblot demonstrating that C. albicans infection induces phosphorylation of Stat3. ( b ) Effects of the EphA2 depletion with siRNA on C. albicans -induced phosphorylation of Stat3. ( c ) Time course of Stat3 phosphorylation induced by zymosan. Densitometric analyses of replicate immunoblots are shown in Supplementary Fig. 19 . ( d ) Oral epithelial cells were incubated with inhibitors of EphA2 and Stat3 and then infected with C. albicans for 5 h, after which chemokines and the S100a8 alarmin mRNA levels were determined by real-time PCR. Box whisker plots show median and range of 2 experiments, each performed in triplicate and are presented as fold induction relative to uninfected epithelial cells. ( e ) EphA2 depletion with siRNA reduces epithelial cell production of human β defensin 2, cytokines and chemokines in response to 8 h of C. albicans infection. Box whisker plots show median and range of 3 experiments, each performed in duplicate. ( f ) Regulation of the oral epithelial cell pro-inflammatory response to C. albicans by Stat3 and dectin-1. Box whisker plots show median and range of 3 experiments, each performed in duplicate. ANT, EphA2 antagonist; ctrl, control; DAS, dasatinib; hBD2, human β defensin 2; MOI, multiplicity of infection; Stat3 INH, Stat3 inhibitor; UNINF, uninfected. ** P < 0.01, *** P < 0.001, **** P < 0.0001; (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Western Blot, Infection, Phospho-proteomics, Incubation, Real-time Polymerase Chain Reaction, Whisker Assay, Control, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Immunoblots showing the effects of increasing C. albicans inoculum on the extent of EphA2 phosphorylation. Densitometric analyses of replicate immunoblots are shown in . ( b ) Effects of C. albicans inoculum on epithelial cell secretion of IL-8 and hBD2. Box whisker plots show median and range of 3 independent experiments in duplicates. ctrl, control; hBD2, human β defensin 2; MOI, multiplicity of infection; Stat3 INH, Stat3 inhibitor; UNINF, uninfected. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Western Blot, Phospho-proteomics, Whisker Assay, Control, Infection, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Oral fungal burden of immunocompetent wild-type and EphA2 −/− mice with oropharyngeal candidiasis after 1 d of infection. Results are median ± interquartile range of 4 mice per group in a single experiment. * P < 0.05 (Mann-Whitney Test) ( b ) Level of chemokines and cytokines in the tongue homogenates of immunocompetent wild-type and EphA2 −/− mice with OPC after 1 d of infection. Box whisker plots show median and range of 4 mice in each group, tested in duplicate in a single experiment. ( c ) Body weight wild-type and EphA2 −/− mice that were immunosuppressed with triamcinolone (7 mg/kg) prior to oral infection with C. albicans . Results are mean ± SD. * P < 0.05 (Holm-Sidak method). ( d ) Oral fungal burden of triamcinolone treated (7 mg/kg) wild-type and EphA2 −/− mice after 4 d of infection ( e–h ). Wild-type mice were immunosuppressed with triamcinolone (15mg/kg), treated with either DAS or the vehicle control, and orally inoculated with C. albicans . They were analyzed after 4 d of infection. ( e ) Oral fungal burden. Results are the median ± interquartile range of combined data from 2 independent experiments for a total of 14 mice in the control group and 13 mice in the DAS group. **** P < 0.0001 (Mann-Whitney Test) ( f ) Liver fungal burden. Results are median ± interquartile range of 7 mice per group in a single experiment. ** P < 0.01 (Mann-Whitney Test) ( g ) Immunohistochemistry of the tongue of a control mouse showing that β-glucan is expressed on the fungal cell surface during oropharyngeal candidiasis. The tongue was stained with Fc-Dectin-1 (green), an anti- Candida antibody (red), and DAPI (blue) Scale bar 100 μm. Results are representative of 3 mice from the same experiment. ( h ) mRNA expression of the indicated inflammatory mediators in the mouse tongue after 4 d of infection. mRNA levels were determined by ΔΔ CT method and normalized to GAPDH. Results are presented as fold change relative to the vehicle control mice. Data from two mice (M1 and M2) are presented. DAS, dasatinib; Veh, vehicle; WT, wild type.

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Infection, MANN-WHITNEY, Whisker Assay, Control, Immunohistochemistry, Staining, Expressing

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( Left panel ) EphA2 (blue) and dectin-1 (green) bind to exposed β-glucan on the fungal surface. Binding to EphA2 is independent of Ca 2+ and activates mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (Stat3). Binding to dectin-1 requires Ca 2+ and activates nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). ( Right panel ) During fungal proliferation, prolonged activation of EphA2 via EGFR (grey) induces the endocytosis of C. albicans and a pro-inflammatory response in the epithelial cells. EphA2-EGFR induces endocytosis and triggers MAPK signaling. EphA2 also activates Stat3 Activation of the Stat3 and MAPK pathways leads to the release of alarmins, cytokines, chemokines, and host defense peptides (HDPs, orange helix).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml −1 ) for 10, 20 and 60 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. ( b ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. ( c ) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml −1 ) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. ( d ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. ( e ) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page, Staining

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HeLa ( a ) or T98G ( b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min ( b , right). ( c ) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. ( d ) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. ( e ) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, shRNA, Expressing, Luciferase, Negative Control

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. ( c ) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. ( d ) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Negative Control, Recombinant, Incubation, Western Blot

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. ( b – f ) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin ( b ) Migrated cells were counted manually under a microscope ( c ) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. At the same time, the migration border cells were immunofluorescently stained with anti-pS-EphA2 or EphA2 antibodies ( d , e ) and cells harbouring lamellipodia were counted manually under a microscope ( f ) Scale bar, 20 μm. Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. ( g , h ) MDA-MB-231 cells were transfected with siRNA against EphA2 or negative control and EphA2 mutation-expression plasmids. The immunoblotting results from whole-cell lysates with anti-pS-EphA2, EphA2 and β-actin antibodies are shown in g and the results of scratch assay are shown in h . Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by analysis of variance followed by Tukey–Kramer HSD test.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page, Transferring, Incubation, Microscopy, Migration, Staining, Transfection, Negative Control, Mutagenesis, Expressing, Western Blot, Wound Healing Assay

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Human melanoma cells (A2058, SK-MEL-28, A375, UACC62, UACC257 and SK-MEL-2), ( b ) DLD-1 colon cancer cells and ( c ) lung adenocarcinoma cells (PC-9, HCC827, HCC4006, NCI-H1650, H2228 and A549) were treated with vemurafenib (1 μM), BI-D1870 (10 μM), gefitinib (1 μM), crizotinib (10 μM) or U0126 (5 μM) for 30–60 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pY-EGFR, EGFR, β-actin and α-tubulin.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) A multi-cancer tissue microarray, including 1,010 cores from 13 organ cancer tissues, was adopted for immunohistochemical staining using primary antibodies against pS-EphA2 and pRSK. Typical staining images of lung cancer tissues, including adenocarcinoma (AD) and squamous cell carcinoma (SCC), at low- and high-power magnifications are shown. Scale bar, 20 μm. ( b ) Typical immunohistochemical staining of pS-EphA2 and pRSK in EGFR -mutated (exon 19 deletion) lung adenocarcinoma tissues are shown. Scale bar, 20 μm. ( c – f ) Postoperative overall Kaplan–Meier survival curves of all the lung cancer patients ( c , d ) or smoking patients ( e , f ) were compared according to pRSK negativity or positivity ( c , e ) or pS-EphA2/pRSK double positivity ( d , f ) P values were calculated by the log-rank tests.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Microarray, Immunohistochemical staining, Staining

Journal: International Journal of Women's Health

Article Title: Molecular Mechanisms Behind Vascular Mimicry as the Target for Improved Breast Cancer Management

doi: 10.2147/IJWH.S406327

Figure Lengend Snippet: Primer Sequences for Real-Time PCR Used in the Study

Article Snippet: After deparaffinization, dehydration, antigen retrieval and blocking endogenous peroxidase, the sections were incubated with primary antibodies against EphA2 (1:500, ZENBIO, 220092), CTNNB1 (1:500, ZENBIO, R22820) and PIK3R1 (1:500, Bioss, Bsm-33219M) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: International Journal of Women's Health

Article Title: Molecular Mechanisms Behind Vascular Mimicry as the Target for Improved Breast Cancer Management

doi: 10.2147/IJWH.S406327

Figure Lengend Snippet: Expression level of the prognostic value in normal tissues and breast cancer samples from the TCGA Database. ( A ) EPHA2, ( B ) PIK3R1, ( C ) CTNNB1, ( D ) PECAM1.

Article Snippet: After deparaffinization, dehydration, antigen retrieval and blocking endogenous peroxidase, the sections were incubated with primary antibodies against EphA2 (1:500, ZENBIO, 220092), CTNNB1 (1:500, ZENBIO, R22820) and PIK3R1 (1:500, Bioss, Bsm-33219M) at 4°C overnight.

Techniques: Expressing

Journal: International Journal of Women's Health

Article Title: Molecular Mechanisms Behind Vascular Mimicry as the Target for Improved Breast Cancer Management

doi: 10.2147/IJWH.S406327

Figure Lengend Snippet: Survival curves comparing low and high expressions of EphA2/PIK3R1/CTNNB1/PECAM1 in breast cancer. ( A ) EPHA2, ( B ) PIK3R1, ( C ) CTNNB1, ( D ) PECAM1. All the survival analyses were conducted via Kaplan–Meier Plotter database ( https://kmplot.com/analysis ).

Article Snippet: After deparaffinization, dehydration, antigen retrieval and blocking endogenous peroxidase, the sections were incubated with primary antibodies against EphA2 (1:500, ZENBIO, 220092), CTNNB1 (1:500, ZENBIO, R22820) and PIK3R1 (1:500, Bioss, Bsm-33219M) at 4°C overnight.

Techniques:

Journal: International Journal of Women's Health

Article Title: Molecular Mechanisms Behind Vascular Mimicry as the Target for Improved Breast Cancer Management

doi: 10.2147/IJWH.S406327

Figure Lengend Snippet: Expression level and correlation analysis of studied genes in breast cancer tissue. ( A – C ) mRNA expression of EphA2/PIK3R1/CTNNB1 by qRT-PCR assay, n=20, *Denotes P <0.05 compared with paracancerous group. ( D – F ) Pearson correlation analysis of EphA2/PIK3R1/CTNNB1/ PECAM1 in breast cancer samples from TCGA database. ( G ) The correlation of EPHA2 and PIK3R1 in breast cancer samples, n=20, R 2 =0.4257, P =0.0018. ( H ) The correlation of PIK3R1and CTNNB1 in breast cancer samples, n=20, R 2 =0.2215, P =0.0362. ( I ) The correlation of EPHA2 and CTNNB1 in breast cancer samples, n=20, R 2 =0.2279, P =0.0333.

Article Snippet: After deparaffinization, dehydration, antigen retrieval and blocking endogenous peroxidase, the sections were incubated with primary antibodies against EphA2 (1:500, ZENBIO, 220092), CTNNB1 (1:500, ZENBIO, R22820) and PIK3R1 (1:500, Bioss, Bsm-33219M) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Women's Health

Article Title: Molecular Mechanisms Behind Vascular Mimicry as the Target for Improved Breast Cancer Management

doi: 10.2147/IJWH.S406327

Figure Lengend Snippet: Immunohistochemical staining of studied genes in para-carcinoma and breast cancer tissues. ( A ) Expression intensity and location of EphA2 in para-carcinoma tissue and breast cancer tissue by immunohistochemical staining assay. ( B ) Quantitative analysis of EphA2 from immunohistochemical images by histochemical scores. ( C ) Expression intensity and location of PIK3R1 in para-carcinoma tissue and breast cancer tissue by immunohistochemical staining assay. ( D ) Quantitative analysis of PIK3R1 from immunohistochemical images by histochemical scores. ( E ) Expression intensity and location of CTNNB1 in para-carcinoma tissue and breast cancer tissue by Immunohistochemical staining assay. ( F ) Quantitative analysis of CTNNB1 from immunohistochemical images by histochemical scores. n=20, *Denotes P <0.05 compared with paracancerous group.

Article Snippet: After deparaffinization, dehydration, antigen retrieval and blocking endogenous peroxidase, the sections were incubated with primary antibodies against EphA2 (1:500, ZENBIO, 220092), CTNNB1 (1:500, ZENBIO, R22820) and PIK3R1 (1:500, Bioss, Bsm-33219M) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Expressing