Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: Primer sequences for qRT-PCR

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques:

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of the lentivirus-mediated shRNA interference of TNFR2 on TNF-α-stimulated EphB4 expression and osteogenic differentiation. a MC3T3-E1 cells stably transduced with lentiviral particles were selected with puromycin and named as pHBLV-TNFR2siRNA1 cells, pHBLV-TNFR2siRNA2 cells, pHBLV-TNFR2siRNA3 cells and pHBLV-NC cells, respectively. The mRNA levels of Tnfr2 were determined in these cells, among which the pHBLV-TNFR2siRNA1 cells displayed the highest TNFR2 gene silencing efficiency and were selected to continue the following studies. b TNFR2 protein levels in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells. c , d mRNA levels of Ephb4 , Runx2 and Bsp in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells cultured in the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α for 24 h ( c ) or 48 h ( d ). e , f Protein levels of EphB4, RUNX2 and BSP in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells cultured in the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α for 24 h ( e ) or 48 h ( f ). *, p < 0.05 vs. the pHBLV-NC group; **, p < 0.01 vs. the pHBLV-NC group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: shRNA, Expressing, Stable Transfection, Transduction, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of the impaired binding between TNF-α and TNFR2 on TNF-α-stimulated EphB4 expression and osteogenic differentiation. MC3T3-E1 cells were treated with an anti-mouse TNFR2/ CD120b/TNFRSF1B neutralizing antibody (TNFR2 NAb) at the concentration of 0.2 μg/ml, and were cultured in the osteogenic induction medium supplemented with or without 0.5 ng/ml TNF-α. Cells treated with 0.2 μg/ml of the normal rabbit IgG negative control antibody (control Ab) served as negative controls. (a) ALP activities were determined 7d or 14d after the treatment. (b, c) mRNA levels of Ephb4 , Runx2 and Bsp were determined after 24 h (b) or 48 h (c). (d, e) Protein levels of EphB4, RUNX2 and BSP were determined after 24 h (d) or 48 h (e). a, p < 0.05 vs. the control Ab group; b, p < 0.05 vs. the TNFR2 NAb group; c, p < 0.05 vs. the TNF-α + control Ab group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Binding Assay, Expressing, Concentration Assay, Cell Culture, Negative Control

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of inhibited EphB4 forward signaling on TNF-α-stimulated TNFR2 expression and osteogenic differentiation. (a) A potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, was used to suppress EphB4 forward signaling. MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 in the regular culture medium for 1 h. Cells were then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α for 7d or 14d. MC3T3-E1 cells cultured in osteogenic induction medium served as controls. The ALP activities were determined. (b, c) MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 for 1 h in the regular culture medium, and then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α. Cells cultured in osteogenic induction medium served as controls. mRNA levels of Tnfr2 , Runx2 and Bsp were determined after 24 h (b) or 48 h (c) of incubation. (d, e) MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 for 1 h in the regular culture medium, and then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α. Cells cultured in osteogenic induction medium served as controls. Protein levels of TNFR2, RUNX2 and BSP were determined after 24 h (d) or 48 h (e) of incubation. a, p < 0.05 vs. the control group; b, p < 0.05 vs. the NVP-BHG712 group; c, p < 0.05 vs. the TNF-α group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Expressing, Incubation, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: EphB4, TNFR2 and MAPK signaling pathways comprise a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation. a Levels of p38, p -p38, ERK1/2, p -ERK1/2, JNK1 + 2 + 3 and p -JNK1 + 2 + 3 in MC3T3-E1 cells treated with TNF-α for 0 min, 5 min, 15 min, 30 min and 60 min. b Levels of p38, p -p38, ERK1/2, p -ERK1/2, JNK1 + 2 + 3 and p -JNK1 + 2 + 3 in the pHBLV-TNFR2siRNA1 cells and the pHBLV-NC cells treated with or without 0.5 ng/ml TNF-α in regular culture medium for 15 min. c MC3T3-E1 cells were pretreated with or without 200 nM NVP-BHG712 in the regular culture medium for 1 h, and then 0.5 ng/ml TNF-α was added into the medium. The cells were incubated for another 15 min. Levels of ERK1/2 and p -ERK1/2 were determined. d-f MC3T3-E1 cells were cultured in the regular culture medium and pretreated with the ERK inhibitor U0126 (10 μM) for 1 h. The culture medium was then switched to the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α and U0126 (10 μM). Cells treated without U0126 (10 μM) served as controls. ALP activities were determined 7d or 14d after the treatment ( d ). mRNA levels ( e ) and protein levels ( f ) of BSP and RUNX2 were determined 3 days after the treatment. *, p < 0.05 vs. the control group; **, p < 0.01 vs. the control group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Incubation, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: Schematic diagram of the EphB4, TNFR2 and ERK/MAPK signaling pathways. A low concentration of TNF-α first enhances the expression of EphB4, which in turn promoted the expression of TNFR2. The elevated TNFR2 level leads to the activation of the ERK signaling pathway, which eventually enhances the osteogenic differentiation of MC3T3-E1 cells. TNFR2, tumor necrosis factor receptor 2; TNF-α, tumor necrosis factor-alpha; MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Concentration Assay, Expressing, Activation Assay

Journal: Journal of Cellular Biochemistry

Article Title: EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

doi: 10.1002/jcb.25000

Figure Lengend Snippet: Quantitative analysis of gene expression by real‐time PCR. GFP + cells at day‐8 EBs from Nkx2.5‐GFP ES cells (Nkx2.5 + ), and GFP + cells at day‐13 EBs from α‐MHC‐GFP ES cells (αMHC + ) were sorted by FACS. Total RNA was extracted from sorted GFP + cells with TRIzol reagent, and subject to real‐time PCR analysis for the gene expression of Flk1 (A), Nkx2.5 (B), GATA4 (C), ephrinB2 (D), and EphB4 (E). Undifferentiated ES cells (ESC) were used as a control. GAPDH was used as a standard. Error bars represent standard deviation and * represents P < 0.05.

Article Snippet: Western blot analyses were performed with anti‐EphB4 antibodies (BD Biosciences) as primary antibodies and anti‐rabbit IgG‐HRP antibodies (Sigma–Aldrich) as secondary antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Cellular Biochemistry

Article Title: EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

doi: 10.1002/jcb.25000

Figure Lengend Snippet: Impairing cardiac differentiation by blocking the interaction of EphB4 and ephrinB2 with TNYL‐RAW peptide. α‐MHC‐GFP ES cells were differentiated into beating EBs in the presence of TNYL‐RAW peptide (TNYLFSPNGPIARAW). The peptides were added at day 0, and through the end of differentiation. The frequency of beating EBs (A) and GFP + cells (B) were analyzed at day 12. (C) The addition of TNYL‐RAW peptides was initialized at different time points during ES cell differentiation: day 0 (EB0), day 2 (EB2), day 5 (EB5), or day 9 (EB9). ES cell differentiation was compared in the presence of the control peptide as added starting at day 0. Data are representative from three independent experiments. Error bars represent standard deviation and * represents P < 0.05.

Article Snippet: Western blot analyses were performed with anti‐EphB4 antibodies (BD Biosciences) as primary antibodies and anti‐rabbit IgG‐HRP antibodies (Sigma–Aldrich) as secondary antibodies.

Techniques: Blocking Assay, Cell Differentiation, Standard Deviation

Journal: Journal of Cellular Biochemistry

Article Title: EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

doi: 10.1002/jcb.25000

Figure Lengend Snippet: Rescue of cardiomyocyte‐defect in EphB4‐null ES cells by ectopic expression of human EphB4 gene. The full‐length of human EphB4 cDNA was used to express human EphB4 gene in EphB4‐null ES cells (KO) by a lentiviral vector to establish ES cell line expressing human EphB4 (KO‐hB4). The expression of EphB4 was analyzed by real‐time PCR (A), Western blotting (B), and flow cytometry using anti‐human EphB4 antibodies (C). (D) Analysis of beating EBs. ES cells of KO, KO‐hB4, and mixtures of KO‐hB4 ES cells with KO ES cells at different ratios (1:1, 1:3, 1:5, and 1:9) were induced for cardiomyocyte differentiation. The frequency of beating EBs was determined at day 12. Data are representative from three independent experiments. Error bars represent standard deviation and * represents P < 0.05.

Article Snippet: Western blot analyses were performed with anti‐EphB4 antibodies (BD Biosciences) as primary antibodies and anti‐rabbit IgG‐HRP antibodies (Sigma–Aldrich) as secondary antibodies.

Techniques: Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Standard Deviation

Journal: Journal of Cellular Biochemistry

Article Title: EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

doi: 10.1002/jcb.25000

Figure Lengend Snippet: Requirement of EphB4‐forward signaling in cardiac differentiation of ES cells. (A) Schematic diagram of an inducible lentiviral system. (B) Western blotting analysis human EphB4 expression induced by doxycycline (Dox) at different concentrations. (C) Schematic diagram of human EphB4 protein with a structure of truncated intracellular domain, including kinase domain and PDZ domain (ΔhB4). (D) Analysis of beating EBs. EphB4‐null ES cells (KO), EphB4‐null ES cells expressing human EphB4 (KO‐hB4), EphB4‐null ES cells expressing truncated EphB4 (KO‐ΔhB4), and wild‐type ES cells (Ctrl) were differentiated to cardiomyocytes. The frequency of beating EBs was analyzed at different time points.

Article Snippet: Western blot analyses were performed with anti‐EphB4 antibodies (BD Biosciences) as primary antibodies and anti‐rabbit IgG‐HRP antibodies (Sigma–Aldrich) as secondary antibodies.

Techniques: Western Blot, Expressing

Journal: Journal of Translational Internal Medicine

Article Title: Effect of local anti-vascular endothelial growth factor therapy to prevent the formation of stenosis in outflow vein in arteriovenous fistula

doi: 10.2478/jtim-2021-0045

Figure Lengend Snippet: (A) The images under confocal microscopy in immunofluorescence studies of representative sections of tissues from the outflow vein in patients of stenosis and control groups. The first column shows DAPI intensity indicating blue-stained nuclei. The second column shows GFP intensity. Magenta-stained cells are GFP positive. The third column depicts merged images. These sections demonstrate different levels of expression of the VEGF protein in the outflow vein between the two groups. (B) The expression of vascular tissue VEGF-A and Ephb4 protein levels in patients. VEGF and Ephb4 were more highly expressed in the AVF group ( P < 0.05). (C) Expression of VEGF-A and Ephb4 in mouse models. Compared to the sham group, the level of expression of VEGF-A and Ephb4 in the control-shRNA group was significantly higher ( P < 0.05). VEGF-A expression in the Avastin group was lower than in the control-shRNA group ( P < 0.05), and the mean levels of Ephb4 protein in the outflow vein of the LV-shRNA-VEGF-A group and Avastin group were significantly lower ( P < 0.05). *Indicates a significant difference compared with the sham group ( P < 0.05). #Indicates a significant difference compared with the control-shRNA group ( P < 0.05). Values are shown as means ± SD from three independent experiments. Representative images are shown from a total of four animals per group.

Article Snippet: After being transferred to PVDF membranes, the membranes were blocked with 5% skimmed milk and incubated with primary antibody: rabbit polyclonal antibody VEGF-A (Abcam, 1:600, ab1316), rabbit anti-EphB4 (Immunoway, 1:600, YT1587), rabbit anti-TIMP1 (Immunoway, 1:600, YT4658), rabbit anti-TIMP2 (Immunoway, 1:600, YT4659), and rabbit anti-caspase 3 (Immunoway, 1:600, ab2302), probing separately.

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Expressing, shRNA

Journal: Journal of Translational Internal Medicine

Article Title: Effect of local anti-vascular endothelial growth factor therapy to prevent the formation of stenosis in outflow vein in arteriovenous fistula

doi: 10.2478/jtim-2021-0045

Figure Lengend Snippet: (A) Bioinformatics network of interaction among VEGF-A and its downstream-regulated molecules MMP2, TIMP1, TIMP2, and EphB4 by analyzing STRING PPI database. (B) MMP2, MMP9, TIMP1, and TIMP2 protein expression levels in the tissue of outflow veins in patients of different groups. MMP2, MMP9, TIMP1, and TIMP2 were higher in the AVF groups. (C) MMP2, MMP9, TIMP1, and TIMP2 in the four mice groups. The level of MMP9 expression was lower in the Avastin and LV-shRNA groups compared with the control-shRNA group on day 14 ( P < 0.05), and the level of MMP2 expression was lower in the Avastin group compared with the control-shRNA group ( P < 0.05). The expression of TIMP1 and TIMP2 in the outflow vein was significantly lower in the Avastin-treated group and LV-shRNA–transduced group compared with the control-shRNA group ( P < 0.05). Also, there was a substantial difference in TIMP2 expression between Avastin-treated and LV-shRNA groups ( P < 0.05). *Indicates a significant difference compared with the sham group ( P < 0.05). #Indicates a significant difference compared with the control-shRNA group ( P < 0.05). &indicates a significant difference compared with the LV-shRNA-VEGF group. Values are shown as means ± SD from three independent experiments. Representative images are shown from a total of four animals per group.

Article Snippet: After being transferred to PVDF membranes, the membranes were blocked with 5% skimmed milk and incubated with primary antibody: rabbit polyclonal antibody VEGF-A (Abcam, 1:600, ab1316), rabbit anti-EphB4 (Immunoway, 1:600, YT1587), rabbit anti-TIMP1 (Immunoway, 1:600, YT4658), rabbit anti-TIMP2 (Immunoway, 1:600, YT4659), and rabbit anti-caspase 3 (Immunoway, 1:600, ab2302), probing separately.

Techniques: Expressing, shRNA