Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic representation of sample preparation and workflow for SiMPull-POP. 1-Membrane fractions containing EphA2-GFP were solubilized with the amphipathic copolymer DIBMA to generate DIBMALPs. 2-EphA2-GFP DIBMALPs were immobilized on a functionalized microscope slide displaying an EphA2 antibody. 3-DIBMALPs devoid of EphA2-GFP are washed away before imaging. (B) Representative single-molecule TIRF image in the presence (left) and absence (right) of EphA2 antibody. Each blue spot represents a DIBMALP containing EphA2-GFP. (C) Representative GFP photobleaching traces showing a stepwise decrease in GFP intensity over time; arrows represent individual photobleaching events. Photobleaching steps are used to infer EphA2 oligomerization status.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Sample Prep, Membrane, Microscopy, Imaging

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: Western blot analysis of EphA2 pS897 in HEK293T (A) , A375 (B) and A431 (C) cells. We show pS897 quantification (mean ± S.D) and representative blots. p -values from one-way ANOVA followed by Mann-Whitney U or t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) cAMP quantification in HEK293T cells transduced with Red Up cADDis cAMP biosensor ollowing treatment with MβCD. (B) Western blot analysis and quantification of PKA T197 phosphorylation in A375 cells following treatment with MβCD. (C-D) Western blot analysis and quantification of EphA2 S897 phosphorylation in A375 cells following treatment with forskolin and soproterenol, respectively. Data shown in panel B are normalized to the respective total PKA signal in Figure S15A. Data shown in panels C-D are normalized to the respective total EphA2 signal in Figure S15B-C. Quantitative comparisons between treatments were made with respect to normalized control conditions. Bar graphs show mean ± S.D., p -values in panels A-D are from an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Transduction, Western Blot, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) In the presence of normal levels of Chol (yellow oval), EphA2 (blue) can be found as a monomer and displays low Ser897 phosphorylation. We propose that in this state the kinase domain interacts with the SAM domain. (B) When Chol content is reduced, β-AR (purple) activity increases, promoting cAMP/PKA signaling that enhances Ser897 phosphorylation (red dot). This forces the kinase-SAM linker into an open conformation, which promotes higher-order oligomers independent from ligand stimulation.

Article Snippet: To immobilize protein samples, 0.02 mg/mL NeutrAvidin protein (Thermo Fisher Scientific) was first incubated in the chamber for 10 minutes followed by the addition of a biotinylated EphA2 or GFP antibody for 20 minutes (Cell Signaling and Rockland Immunochemical Inc., respectively).

Techniques: Phospho-proteomics, Activity Assay

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–C) Epha2 homozygous deletion in mice causes development of progressive cataract. (A) Cataracts were visible by gross inspection in homozygous Epha2 knockout mice ( Epha2 −/− ) between 5 to 8 months of age, but not in heterozygous or wild type mice. Shown are slit lamp images confirming development of cataract in Epha2 −/− but not Epha2 +/+ mice. (B) Dark field imaging of the dissected lens. Although not readily detectable by visual inspection, cataracts were found on dissected lens by 3 months of age. This lens was tilted to show denser opacity near the equator (arrow). Enucleation frequently occurred during dissection of mature cataract after 8 months (far right). (C) Retroillumination examination revealed clusters of small vacuoles by one month of age. Scale bars: 1 mm for middle panel; 150 µm for right panel. (D) Immunoblot of total lens lysates showing decreasing EPHA2 expression with aging. (E–M) Compartmentalized and gradient expression of EPHA2 (red) in mouse lens. Blue: DAPI nuclear staining. (E–I) Midsagittal sections of lens from 14-day-old wild type mice were stained for EPHA2. (E) Low power view of an entire lens revealed dense expression of EPHA2 in subcortical lens fiber cells. Dotted arrows indicate gradient expression in lens epithelial cells near the equator. Scale bar: 1 mm. (F) Low EPHA2 expression in anterior lens epithelial cells (arrow head, sandwiched between dotted lines). (G) Inset from (F) showing high EPHA2 expression in lens fiber cells. (H) High level of EPHA2 expression at the bow. (I) Inset from (H) showing dense expression at modulus (arrow). Scale bars: 5 µm for F–I. (J–M) Coronal sections through the bow region of lens co-stained for EPHA2 and N-cadherin. (J) Note the spatially regulated expression pattern in subcortical lens fiber cells. (K) Inset from (J) showing “honey-comb” membrane staining pattern of EPHA2 in the cross sections of fiber cells at high magnifications. (L) N-cadherin from the same section show overlapping but distinct expression pattern compared with that of EPHA2. (M) Merged images of EPHA2/N-cadherin. 10 µm for J, L, and M; 2 µm for K.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Knock-Out, Imaging, Dissection, Western Blot, Expressing, Staining, Membrane

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Incidence of Visible Cataracts in Wild-Type and Epha2 -null Mice.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–D) Expression of ephrin-A1, a ligand for EPHA2. Note the disorganized ephrin-A1 expression and formation of vacuoles in the Epha2 −/− lens (arrow heads). Scale bars: 1 mm for A and C; 10 µm for B and D. (E,F) N-cadherin staining showing disorganization of lens fiber cells. Scale bars: 5 µm. (G) Overexpression of HSP25 but not HSP90 in Epha2 −/− lens which was quantified in (H), and confirmed by immunofluorescence staining (I). Scale bars: 40 µm. (J) Immunoblot for phosphorylated HSP25 revealed relatively low degree of phosphorylation in Epha2 −/− lens.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Expressing, Staining, Over Expression, Immunofluorescence, Western Blot, Phospho-proteomics

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Characteristics of SNPs in the EPHA2 gene.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: P values from rank transformed traits and effect sizes from the quantitative cortical scores (β) of the risk allele at markers under the dominant model in EPHA2 for each separate study and for the joint analysis of all studies.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Transformation Assay

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A) Examination of crystal structure of EPHA2 kinase domain reveals that Arg721 in αE forms a salt bridge with Asp872 in αI. (B) Concordant conservation of Arg721 and Asp872 in different members of human Eph kinases. Note that EPHA9 and EPHB5 are not present in human genome and are not shown. (C) The same residues are also concordantly conserved across different species.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A,B) Arg721Gln mutation causes an increased basal activation of EPHA2 kinase, which was correlated with dramatically reduced basal ERK1/2 activities. In a kinetic study (A), HEK 293 cells expressing WT-, Arg721Gln -EPHA2 or vector control were stimulated with 2 µg/ml ephrin-A1-Fc for the indicated times. In a separate experiment, a dose-response study was carried out (B), where different doses of ephrin-A1-Fc were used to stimulate cells expressing WT- or Arg721Gln -EPHA2 for 10 min. Cell lysates from both experiments were blotted with the indicated antibodies as described previously . (C) HEK 293 cells expressing Arg721-Gln mutant EPHA2 but not WT-EPHA2 were growth-inhibited by ephrin-A1 in a clonal growth assay as described previously . About 200 cells/well were seeded in a 24-well culture dish and cultured for 10 days in the presence or absence of ephrin-A1. (D) Stochastic intracellular trapping of Arg721Gln mutant in MEF cells derived from Epha2 knockout embryos. Shown is a cluster of cells with the mutant EPHA2 trapped inside the cells. In contract, WT-EPHA2 was primarily expressed on the cytoplasmic membrane. Scale bar: 5 µm.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Control, Growth Assay, Cell Culture, Derivative Assay, Knock-Out, Membrane

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a IHC showing the levels of HDAC7 and EphA2 in the NPCs and normal nasopharyngeal mucosa (NNM). Representative IHC images are shown on the top, and statistical analysis is presented on the bottom ( p < 0.001, Chi-squared test). b Western blot showing the levels of HDAC7 and EphA2 in the additional four paired fresh biopsies of NPC and NNM. c Western blot showing the levels of HDAC7 in the five NPC cell lines and immortalized normal human nasopharynx epithelial cell line NP69. d Kaplan–Meier survival analysis for 107 NPC patients according to HDAC7 expression levels. NPC patients with high HDAC7 expression have a significantly worse overall survival and disease-free survival than those with low HDAC7 expression. The log-rank test was used to calculate P value.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Western blot and qRT-PCR showing the levels of EphA2 protein and mRNA in the shHDAC7 HK1 and 5–8F cells and their control cells. b Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells and control cells treated by 50 μM chloroquine (CQ) and 20 μM MG132, respectively. c QRT-PCR showing the levels of EphA2 hnRNAs in the shHDAC7 HK1 and 5–8F cells and control cells. d Western blot showing the levels of EphA2 in the HK1 and 5–8F cells transfected with 100 nM miR-4465 mimic or mimic control. e QRT-PCR showing the levels of miR-4465 in the shHDAC7 HK1 and 5–8F cells and control cells. f Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells transfected with 200 nM miR-4465 inhibitor or inhibitor control. g 3′UTR dual-luciferase reporter assay. (Top) The predicted miR-4465 binding sites in the 3′UTR of wild-type (WT) EphA2 and mutant EphA2 3′UTR are shown; (bottom) luciferase activity of WT and mutant EphA2 3′UTR dual-luciferase reporter vector in the HEK293 cells transfected with control or miR-4465 mimic. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Quantitative RT-PCR, Control, Transfection, Luciferase, Reporter Assay, Binding Assay, Mutagenesis, Activity Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a QRT-PCR analysis of the expression levels of miR-4465 in the 107 NPC tissues and 20 NNM. b Western blot showing the levels of EphA2 in the HK1 and 5–8F cells transfected with miR-4465 mimic or co-transfected with miR-4465 mimic and EphA2 expression plasmid, and their control cells. CCK-8 ( c ), plate clone formation ( d ), and EdU incorporation ( e ) assay showing the proliferation of HK1 and 5–8F cells transfected with 100 nM miR-4465 mimic or co-transfected with 100 nM miR-4465 mimic and 1μg/ml EphA2 expression plasmid, and their control cells. Scratch wound healing ( f ) and Transwell Matrigel invasion assay ( g ) showing the migration and invasion of HK1 and 5–8F cells 100 nM miR-4465 mimic or co-transfected with 100 nM miR-4465 mimic and 1 μg/ml EphA2 expression plasmid, and their control cells. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Invasion Assay, Migration

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. b–f Restoration of EphA2 expression antagonized the inhibitory effects of HDAC7 knockdown on NPC cell proliferation, and migration and invasion in vitro. CCK-8 ( b ), plate clone formation ( c ), and EdU incorporation ( d ) assay showing the proliferation of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. e Scratch wound healing showing the migration of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. f Transwell Matrigel invasion assay showing the invasion of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Knockdown, Migration, In Vitro, CCK-8 Assay, Invasion Assay

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and EphA2 siRNA (siEphA2), and their control cells. b–f The effects of miR-4465 inhibitor transfection or miR-4465 inhibitor and siEphA2 co-transfection on NPC cell proliferation, and migration and invasion. CCK-8 ( b ), plate clone formation ( c ), and EdU incorporation ( d ) assay showing the proliferation of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. e Scratch wound healing assay showing the migration of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. f Transwell Matrigel invasion assay showing the invasion of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. g Correlation analyses of HDAC7, miR-4465, and EphA2 in 107 NPC based on their expression levels. (Left) Spearman correlation analysis showing the positive correlation between HDAC7 and EphA2. (Right) Spearman correlation analysis showing the negative correlation between miR-4465 and HDAC7, and miR-4465 and EphA2. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Transfection, Control, Cotransfection, Migration, CCK-8 Assay, Wound Healing Assay, Invasion Assay, Expressing