Journal: International Journal of Molecular Medicine

Article Title: Amyloid precursor protein promotes the migration and invasion of breast cancer cells by regulating the MAPK signaling pathway

doi: 10.3892/ijmm.2019.4404

Figure Lengend Snippet: Effects of APP overexpression and silencing on the expression and phosphorylation levels of MAPK signaling pathway-related factors in MDA-MB-231 breast cancer cells. (A) Protein expression and phosphorylation levels of MAPK signaling pathway-related factors following APP overexpression and silencing. (B) mRNA expression level of MAPK signaling pathway-related molecules by RT-qPCR following APP overexpression and silencing. (C) Statistical analysis of the western blot bands. APP overexpression significantly increased the phosphorylation levels of MLK3, MEK4 and JNK3 in MDA-MB-231 breast cancer cells, when compared with the control groups. All experiments were performed in triplicate. Data are presented as the mean ± SD. * P<0.05 vs. control. APP, amyloid precursor protein; MLK3, mixed lineage kinase 3; MEK4, mitogen-activated protein kinase kinase 4; t-, total; p-, phosphorylated; shRNA, short hairpin RNA; MAPK, mitogen-activated protein kinase.

Article Snippet: Rabbit anti-human APP (1:2,000 for western blot analysis; 1:300 for immunohistochemistry; cat. no. 2452S), mouse anti-human E-cadherin (1:2,000; cat. no. 14472), mouse anti-human N-cadherin (1:2,000; cat. no. 14215), mouse anti-human cytokeratin (1:2,000; cat. no. 4545), mouse anti-human vimentin (1:2,000; cat. no. 49636), mouse anti-human MMP-9 (1:2,000; cat. no. 3852), rabbit anti-human MMP-2 (1:2,000; cat. no. 4022), rabbit anti-human MMP-3 (1:2,000; cat. no. 14351) and rabbit anti-human mitogen-activated protein kinase kinase kinase 11 (MLK3) primary antibodies (1:2,000; cat. no. 2817) were purchased from Cell Signaling Technology, Inc. Rabbit anti-human MEK4 (1:2,000; cat. no. ab33912), rabbit anti-human phosphorylated (p)-MEK4 (1:2,000; cat. no. ab131353), rabbit anti-human p-MLK3 (1:2,000; cat. no. ab191530), rabbit anti-human JNK3 (1:2,000; cat. no. ab126591), rabbit anti-human p-JNK3 (1:2,000; cat. no. ab124956) and rabbit anti-human β-actin primary antibodies (1:4,000; cat. no. ab179467), as well as horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5,000; cat. no. ab6721) and goat anti-mouse (1:3,500; cat. no. ab6789) secondary antibodies were purchased from Abcam.

Techniques: Over Expression, Expressing, Phospho-proteomics, Quantitative RT-PCR, Western Blot, Control, shRNA

Journal: Theranostics

Article Title: 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer

doi: 10.7150/thno.35861

Figure Lengend Snippet: ZNF471 inhibited ESCC growth in vivo . (A, B) Images of human ESCC tumor xenografts. (C) Comparative histogram of tumor weights in the two groups (empty vector group vs. stably expressing ZNF471 group) of nude mice. (D) Comparative analyses of tumor growth curve for vector- and ZNF471-infected KYSE150 cells in nude mice xenografts. (E, F) Representative photographs of H&E staining and IHC expression analyses of ZNF471, MAPK10, Ki-67, and apoptosis as assessed by TUNEL assays in xenografts. Scale bars: 100 μm. Student's test was used. Data are presented as the mean ± SD. * p <0.05, *** p <0.001.

Article Snippet: Anti-Flag was obtained from Abm (#G188; Abm, Richmond, BC), anti-MAPK10 was obtained from Bioss (bs-2997R, Bioss, Beijing, China), and anti-Ki-67 was obtained from Abcam (ab15580; Abcam, Cambridge, UK).

Techniques: In Vivo, Plasmid Preparation, Stable Transfection, Expressing, Infection, Staining, TUNEL Assay

Journal: Theranostics

Article Title: 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer

doi: 10.7150/thno.35861

Figure Lengend Snippet: Bioinformatics analysis of possible mechanism of ZNF471 functions in ESCC cells. (A) Ectopic expression of ZNF471 in KYSE150 and KYSE410 cells. (B) Entire distribution of differentially expressed genes in vector- and ZNF471-stably transfected KYSE410 cell is shown by a volcano plot. (C) Hierarchical cluster analysis of selected differentially expressed genes in R software is shown as a heat map (gene functional annotation: PCDH family genes, CLDN1 and CNTN1 involved in cell adhesion, MAPK10/JNK3 and PYCARD involved in cell apoptosis, TUSC3 and SAMD9L involved in tumor suppression, and IFNL3 involved in immunity regulation). (D-G) Validation of differentially expressed genes in KYSE150 and KYSE410 cells ectopically expressing ZNF471 by qRT-PCR. Student's test was used. Data are presented as the mean ± SD. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Anti-Flag was obtained from Abm (#G188; Abm, Richmond, BC), anti-MAPK10 was obtained from Bioss (bs-2997R, Bioss, Beijing, China), and anti-Ki-67 was obtained from Abcam (ab15580; Abcam, Cambridge, UK).

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Software, Functional Assay, Quantitative RT-PCR

Journal: Theranostics

Article Title: 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer

doi: 10.7150/thno.35861

Figure Lengend Snippet: MAPK10 suppresses ESCC cell proliferation and metastasis. (A) Ectopic expression of MAPK10 in KYSE150 and KYSE410 cells. (B) Cell viabilities were evaluated at 24, 48 and 72 h after transfection with MAPK10 in KYSE150 and KYSE410 cells. (C, D) Percentages of apoptotic cells in KYSE150 and KYSE410 cells with MAPK10 ectopic expression were evaluated. Cell apoptosis alterations were revealed by histograms. (E) Evaluation of MAPK10-knockdown in ZNF471-infected ESCC cells after transfection with MAPK10 siRNA and siNC by qRT-PCR. (F) Effects of knockdown of MAPK10 on cell viability, as measured by cell viability assays. (G, H) Percentages of apoptotic cells in ZNF471-infected ESCC cells after MAPK10-knockdown were evaluated. Student's test was used. Data are presented as the mean ± SD. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Anti-Flag was obtained from Abm (#G188; Abm, Richmond, BC), anti-MAPK10 was obtained from Bioss (bs-2997R, Bioss, Beijing, China), and anti-Ki-67 was obtained from Abcam (ab15580; Abcam, Cambridge, UK).

Techniques: Expressing, Transfection, Infection, Quantitative RT-PCR

Journal: Theranostics

Article Title: 19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer

doi: 10.7150/thno.35861

Figure Lengend Snippet: ZNF471 activates MAPK10/JNK3 signaling and downstream proapoptotic activation in ESCC cells. (A) Locations of ChIP PCR primers (segment 1(+9-+136), 2(+116-+136), 3(+261-+419) and 4(+400-+580) at the MAPK10 promoter, transcription start site (TSS) is designated as nucleotide +1.F1, Fragments 1;F2, Fragments 2; F3, Fragments 3; F4, Fragments 4.(B) input % of MAPK10 DNA by anti-Flag antibody were determined by ChIP-qPCR. (C) The effect of ZNF471 on MAPK10/JNK3 signaling, as determined by luciferase reporter activity assays. The pLG3-Tr1 plasmid corresponds to all ChIP primer regions, the pLG3-Tr2 truncated plasmid corresponds to the ChIP primer F1/R1 and F2/R2 regions, the pLG3-Tr3 truncated plasmid corresponds to the ChIP primer F3/R3 region, and the pLG3-Tr4 truncated plasmid corresponds to the ChIP primer F4/R4 region. (D, E) qRT-PCR analysis of MAPK10, caspase 8 and caspase 3. (F) Western blot analysis of MAPK10 and downstream effectors. (G) A schematic diagram of the possible mechanism underlying ZNF471 tumor suppression in ESCC cells. Student's test was used. Data are presented as the mean ± SD. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Anti-Flag was obtained from Abm (#G188; Abm, Richmond, BC), anti-MAPK10 was obtained from Bioss (bs-2997R, Bioss, Beijing, China), and anti-Ki-67 was obtained from Abcam (ab15580; Abcam, Cambridge, UK).

Techniques: Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Pathway analysis of microarray data on genes differentially expressed between control and SH-SY5Y-APP cells. b ChIP-seq screening results in SH-SY5Y cells showed that the region of AICD interaction is in the fourth intron of the JNK3 locus in chromosome 4. TSS, transcription starting site. c Validation of the ChIP-seq results. SH-SY5Y cells were transfected with either pcDNA4/V5-His or pcDNA4/V5-His-hAICD59 plasmid, and subjected to ChIP assay, using normal mouse IgG as control. Input, 5% of the sonicated chromatin. Left panel, real-time PCR results show V5, but not IgG control interacts with the JNK3 intron region ( n = 3). Right panel, representative image shows the real-time PCR products resolved by agarose gel. The molecular weight of the DNA ladder shown in bp on the left. d Real-time PCR results showing the JNK3 mRNA level in pcDNA4/V5-His (control) or pcDNA4/V5-His-hAICD59 (AICD) transfected SH-SY5Y cells ( n = 3). Student’s t- test: ** p < 0.01. Error bars represent SEM. e Luciferase assay showing the enhancer activity of the AICD-interacting site. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Microarray, Control, ChIP-sequencing, Biomarker Discovery, Transfection, Plasmid Preparation, Sonication, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Luciferase, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Intact retinae from wild-type mice were fixed, paraffin-embedded and 5 μm thick longitudinal sections were cut. The sections were stained with anti-JNK3, anti-APP and anti-Tuj1 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. APP and JNK3 were co-expressed in Tuj1 + RGCs. b Intact retinae from wild-type, APP-null or JNK3-null mice were stained with anti-APP or anti-JNK3 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. The anti-APP and anti-JNK3 antibodies were specific as no immunofluorescence signal is seen in the APP-null and JNK3-null retinal sections, respectively. c Adult, wild-type mouse retinae were retrogradely labeled with OHSt, fixed, and 5 μm thick sections were cut. The sections were stained with anti-APP and anti-JNK3 antibodies without antigen retrieval as antigen retrieval destroys the OHSt signal. Arrow heads indicate several OHSt-labeled RGCs, which co-express both APP and JNK3. Scale bar, 20 μm

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Staining, Immunofluorescence, Labeling

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Quantitative analysis of relative mRNA levels of Jnk s in intact retinae or retinae 1 day (1 d), 1 week (1 w) or 2 weeks (2 w) after ONA ( n = 3 for each time point). b Representative western blots from at least three biological repeats showing JNK expression after ONA. Intact retina or retinae 1 d, 1 w, 2 w after ONA were harvested, lysed and immunoblotted using antibodies against JNK1, JNK2, JNK3, pJNK or GAPDH. The location of molecular weight markers is shown in kDa on the right. c Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from wild-type or JNK1, JNK2, or JNK3-null mice ( n = 6 for each genotype) 14 days after ONA using an anti-Tuj1 antibody. d The bar chart shows quantitative analysis of Tuj1 + RGC numbers 14 days after ONA. Scale bar, 50 μm. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Western Blot, Expressing, Molecular Weight, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: Representative western blots show JNK3 expression in a pcDNA3-FLAG-APP695 transfected or pcDNA3 transfected SH-SY5Y cells, e retinae from wild-type or APP-null mice, or i pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. The samples were lysed, resolved in SDS-page gel, and immunoblotted using antibodies against JNK3 or GAPDH. b , f , j The bar charts show quantitative analysis of relative JNK3 expression ( n = 3 for each experiment). Representative western blots show phosphorylative activation of JNK3 in c APP695-overexpressing or normal SH-SY5Y cells, g retinae from wild-type or APP-null mice, or k pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. JNK3 was immunoprecipitated with anti-JNK3 antibody and then detected for kinase activity with anti-phospho-JNK (pJNK) or anti-JNK3 antibody. Whole-cell lysate western blots show the downstream substrate pc-Jun73 or c-Jun level. d , h , l The bar charts show quantitative analysis of activated pJNK/mg lyate proteins or pc-Jun73/c-Jun ratio ( n = 3 for each experiment). Student’s t -test: * p < 0.05; ** p < 0.01. Error bars represent SEM

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Western Blot, Expressing, Transfection, SDS Page, Activation Assay, Immunoprecipitation, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in retinae from wild-type or APP-null mice 1 day (1 d), 1 week (1 w) and 2 weeks (2 w) after ONA ( n = 3 for each time point). Anti-JNK3, anti-pJNK and anti-GAPDH antibodies were used to detect the respective protein expression. b The bar charts show quantitative analysis of relative JNK3 expression or overall phosphorylative activation of JNK in the ipsilateral-injured retinae vs. the contralateral-uninjured retina. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Western Blot, Expressing, Activation Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a – d Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in SH-SY5Y-APP cells treated with vehicle or γ-secretase inhibitors a L-685,458 (5 μM) or c BMS 299897 (1 μM). b , d The bar charts show quantitative analysis of relative JNK3 expression and overall phosphorylative activation of JNK normalized to the GAPDH ( n = 3 for each treatment). e Representative western blots show phosphorylative activation of JNK3 in retinae from vehicle-treated or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA. f Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from vehicle or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA using anti-Tuj1 antibody. Scale bar, 50 μm. g The bar chart shows quantitative analysis of the surviving Tuj1 + RGC number in injured mouse retinae 14 days after ONA ( n = 4 for each treatment). Student’s t -test: ** p < 0.01. Error bars represent SEM

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Western Blot, Expressing, Activation Assay, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: ONA induces upregulation of APP expression. APP is sequentially cleaved by α- or β-secretase and γ-secretase, producing several fragments and AICD, which is inhibited by APP deficiency or BMS 299897. AICD is translocated to the nucleus and upregulates JNK3 expression (probably functioning with other partners). Phosphorylated JNK3 then contributes to RGC death

Article Snippet: The mouse retina or cell lysate was incubated overnight at 4 °C with rabbit anti-JNK3 antibody (Cell Signaling Technology) or normal rabbit IgG control (Cell Signaling Technology), followed by incubation with pre-washed protein G-Sepharose 4 Fast Flow (GE Healthcare) for 3 h at 4 °C.

Techniques: Expressing

Journal: Journal of neurochemistry

Article Title: Akt inhibits MLK3/JNK3 signaling by inactivating Rac1: a protective mechanism against ischemic brain injury.

doi: 10.1111/j.1471-4159.2006.04020.x

Figure Lengend Snippet: Fig. 5 Effects of tyrosine phosphatase inhibition on the interaction of phospho-c- Jun N-terminal kinases (p-JNKs) with 14-3- 3, serine phosphorylation of 14-3-3, inter- action between 14-3-3 and Bax, and mitochondrial translocation of Bax in the hippocampal CA1 region induced by cerebral ischemia–reperfusion. (a) Co- immunoprecipitation and western blotting analysis were used to explore the binding behavior of the interactions of 14-3-3 with phospho-JNK or Bax, and the alteration behavior of serine phosphorylation of 14-3- 3. Homogenates were subjected to immu- noprecipitation with anti-phospho-JNKs or anti-14-3-3 antibody and the immunocom- plexes were probed for the presence of 14- 3-3, Bax and phospho-serine. (c) Effects of pre-treatment with orthovanadate (OV) on the increased phosphorylation of 14-3-3 and the decreased interaction of 14-3-3 with Bax induced by 6 h of ischemia–reperfusion in the hippocampal CA1 region. (d) Effects of tyrosine phosphatase inhibition on the increased Bax translocation to the mito- chondria induced by 6 h of ischemia–rep- erfusion in the hippocampal CA1 region. (b, e) Bands on western blots were scanned and the data were expressed as a per- centage of the value of sham-operated rats. Values are given as the mean ± SD of four independent experiments (n ¼ 4). *p < 0.05 versus sham control. #p < 0.05, significant difference versus the vehicle- treated group. IP, immunoprecipitated; OD, optical density; WB, western blotted.

Article Snippet: Antibodies to PTEN (sc-9145), Rac1 (sc-217), p-Rac1 (Ser71, sc-12924-R), MLK3 (sc-13072), active JNKs (recognizes JNK1, JNK2 and JNK3, sc-6254), Fas-L (sc-6237), Fas (sc1023), Bax (sc-493), 14-3-3 (sc-1019) and phospho-c-Jun (sc-822) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition, Phospho-proteomics, Translocation Assay, Immunoprecipitation, Western Blot, Binding Assay, Control

Journal: Nature communications

Article Title: EphrinB2 controls vessel pruning through STAT1-JNK3 signaling

doi: 10.1038/ncomms7576

Figure Lengend Snippet: JNK3 is expressed in regressing hyaloid vessels and mediates EphrinB2-dependent endothelial cell death. (a) STAT1 silencing augments cell viability in HUVEC expressing eB2-5Y. Results reflect the means±SD from 3 experiments; error bars: ±SD. (b,c) eB2-shRNA augments JNK3 mRNA (b; means±SD from 3 experiments; error bars: ±SD) and protein (c) expression in HUVEC. (d) Transduction of eB2-5Y but not eB2-WT augments JNK3 expression in HUVEC; means±SD from 3 experiments; error bars: ±SD (e) Time-dependent JNK3 and cJUN activation in serum-starved HUVEC. Cell lysates from serum-starved HUVEC were tested at the indicated time-points for kinase activity and content of p-JNK1, p-JNK2 and p-JNK3 after immunoprecipitation with anti-p-JNK antibody. (f) JNK3 transduction reduces HUVEC viability; means±SD from 3 experiments; error bars: ±SD (g) JNK3 silencing improves HUVEC viability compromised by eB2-shRNA and B2-5Y transduction; means±SD from 3-5 experiments; error bars: ±SD (h) JNK3 is detected in p5 and p7 WT hyaloid vessels. Degenerating hyaloid vessels are marked by anti-JNK3 antibody staining (red); p-EphrinB immunostaining (green) is absent from JNK3+ hyaloid vessels; DAPI (blue). Scale bars: 200μm top panels, 50μm bottom panels. (i–k) Immunostaining reveals an inverse relationship between JNK3 and p-EphrinB fluorescence intensity in individual IB4 + cells within p5 and p7 WT hyaloid vessels. Cell distribution based on JNK3 and p-EphrinB2 mean fluorescence intensity; each dot represents 1 cell (i); the bar graphs show (j) the means (n=100–300/group) and range (±SD error bars) of p-EphrinB fluorescence intensity in JNK3 + cells and (k) the geographical distribution within hyaloid vessels of JNK3 + (mean fluorescence intensity ≥3.5)/p-EphrinB + (mean fluorescence intensity ≥30) cells (means±SD; n=4 areas/group); fluorescence intensity values in each region are normalized by DAPI. P values from two-tailed Student t -test: N.S. not significant, * P <0.05, ** P < 0.01, *** P <0.001.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit monoclonal or polyclonal IgG antibodies to: SHP2 (no. 3397; 1:1000), p-VEGFR2 (Tyr 1175 ) (no. 2478; 1:1000); VEGFR2 (no. 2479; 1:1000); p-Erk (Thr 202 /Tyr 204 ) (no. 4370; 1:2000); Erk (p44/p42) (no. 9102; 1:1000); p-EphrinB (Tyr 324/ Tyr 329 ) (no. 3481; 1:1000); cleaved PARP (Asp 214 ), (no. 5625; 1:1000); JNK3 (no. 2305; 1:1000); JAK2 (no. 3230; 1:1000), p-JAK2 (Tyr 1007/1008 ) (no. 3776; 1:1000); STAT1 (no. 9175; 1:1000), p-STAT1 (Tyr 701 ) (no. 9167; 1:1000), p-STAT3 (Tyr 705 ) (no. 9145; 1:2000) (all from Cell Signaling Technology); goat anti-SHP2 (Abcam no. Ab110194; 1:250); rabbit anti-JNK3 (Novus Biologicas no. NBP!-19542; 1:2000); goat anti-actin (Santa Cruz Biotechnology no. sc-1616; 1:).

Techniques: Expressing, shRNA, Transduction, Activation Assay, Activity Assay, Immunoprecipitation, Staining, Immunostaining, Fluorescence, Two Tailed Test

Journal: Nature communications

Article Title: EphrinB2 controls vessel pruning through STAT1-JNK3 signaling

doi: 10.1038/ncomms7576

Figure Lengend Snippet: JNK3 is a target of p-STAT1 regulation. (a) HUVEC-endogenous p-STAT1 induced by serum starvation or IFNγ (10 ng/ml) stimulation specifically binds to JNK3 promoter region. Chromatin IP with p-STAT1 antibodies or control IgG; precipitated DNA was measured by qPCR with specific primers for JNK3 promoters. CM: HUVEC complete medium. Results show means (±SD shown as error bars) from 5 replicates. P values from two-tailed Student t-test: * P <0.05, *** P <0.001. (b) WT EphrinB2 represses JAK2+STAT1-driven JNK3 promoter activity; Gaussia luciferase dual-reporter assay; means (±SD shown as error bars) of 5 experiments. P values from two-tailed Student t-test: *** P <0.001. The JNK3 reporter plasmid was co-transfected in HEK293T cells with expression plasmids for JAK2+STAT1; dominant-negative (DN) JAK2+STAT1; JAK2+STAT1+eB2-WT or JAK2+STAT1+eB2-5Y. (c) Schematic representation of experimental results.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit monoclonal or polyclonal IgG antibodies to: SHP2 (no. 3397; 1:1000), p-VEGFR2 (Tyr 1175 ) (no. 2478; 1:1000); VEGFR2 (no. 2479; 1:1000); p-Erk (Thr 202 /Tyr 204 ) (no. 4370; 1:2000); Erk (p44/p42) (no. 9102; 1:1000); p-EphrinB (Tyr 324/ Tyr 329 ) (no. 3481; 1:1000); cleaved PARP (Asp 214 ), (no. 5625; 1:1000); JNK3 (no. 2305; 1:1000); JAK2 (no. 3230; 1:1000), p-JAK2 (Tyr 1007/1008 ) (no. 3776; 1:1000); STAT1 (no. 9175; 1:1000), p-STAT1 (Tyr 701 ) (no. 9167; 1:1000), p-STAT3 (Tyr 705 ) (no. 9145; 1:2000) (all from Cell Signaling Technology); goat anti-SHP2 (Abcam no. Ab110194; 1:250); rabbit anti-JNK3 (Novus Biologicas no. NBP!-19542; 1:2000); goat anti-actin (Santa Cruz Biotechnology no. sc-1616; 1:).

Techniques: Chromatin Immunoprecipitation, Two Tailed Test, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Expressing, Dominant Negative Mutation

Journal: Nature communications

Article Title: EphrinB2 controls vessel pruning through STAT1-JNK3 signaling

doi: 10.1038/ncomms7576

Figure Lengend Snippet: Phenotypes of JNK3 −/− eyes. (a) JNK3 −/− mice display microphthalmia and abnormal retinal folding. Front and rear view of p5 eyes from JNK3 +/+ and JNK3 −/− mice. Arrows: abnormal retinal folds and pockets. Scale bars: 1mm (b) Measurements of eye and lens size in eye sections of JNK3 +/+ and JNK3 −/− p5 mice. The results reflect the means ±SD; n=3 mice/group; P values from two-tailed Student t -test: **P<0.01 and *** P<0.001. (c) Presence of an abnormal retrolental mass in the vitreous region of JNK3 −/− mice. IB4 staining of eye sections in representative p5 JNK3 +/+ and JNK3 −/− pups; scale bars: 500μm. IB4: red; DAPI: blue. Insets are enlarged in panel d. (d) The retrolental mass in JNK3 −/− mice is stained with IB4 (red), p-EphrinB (green) and DAPI (blue). Arrowheads: p-EphrinB + /IB4 + cells; arrows: p-EphrinB − /IB4 + cells; scale bars: 50μm. (e) Retrolental mass from a JNK3 −/− mouse at p5 contains NG2 + pericytes (cyan) and F4/80 + macrophages (green) within an IB4 + vascular-type structure (red). A littermate JNK +/+ mouse displays no retrolental mass; hyaloid vessels are located on the lens. Images are from sections of the entire eyeball. Scale bars: 50μm. (f) The retrolental mass of JNK3 −/− mice is Cleaved caspase3-negative. Cleaved Caspase3: green; IB4: red; DAPI: blue. Arrowheads: Cleaved Caspase3 + /IB4 + cells; arrows: Cleaved caspase3 − /IB4 + retrolental mass. Scale bars: 50μm. (g) A pathological retrolental mass is detected in the eye of a 16 week-old JNK3 −/− mouse but not in a JNK3 +/+ littermate (scale bars: 1mm); inset is magnified on the right (scale bar: 500μm); tissues were stained with colloidal Comassie G-250. (h) Phase contrast imaging of the unstained or DAPI-stained (blue) retrolental mass shown in g; scale bar: 100μm.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit monoclonal or polyclonal IgG antibodies to: SHP2 (no. 3397; 1:1000), p-VEGFR2 (Tyr 1175 ) (no. 2478; 1:1000); VEGFR2 (no. 2479; 1:1000); p-Erk (Thr 202 /Tyr 204 ) (no. 4370; 1:2000); Erk (p44/p42) (no. 9102; 1:1000); p-EphrinB (Tyr 324/ Tyr 329 ) (no. 3481; 1:1000); cleaved PARP (Asp 214 ), (no. 5625; 1:1000); JNK3 (no. 2305; 1:1000); JAK2 (no. 3230; 1:1000), p-JAK2 (Tyr 1007/1008 ) (no. 3776; 1:1000); STAT1 (no. 9175; 1:1000), p-STAT1 (Tyr 701 ) (no. 9167; 1:1000), p-STAT3 (Tyr 705 ) (no. 9145; 1:2000) (all from Cell Signaling Technology); goat anti-SHP2 (Abcam no. Ab110194; 1:250); rabbit anti-JNK3 (Novus Biologicas no. NBP!-19542; 1:2000); goat anti-actin (Santa Cruz Biotechnology no. sc-1616; 1:).

Techniques: Two Tailed Test, Staining, Imaging

Journal: Nature communications

Article Title: EphrinB2 controls vessel pruning through STAT1-JNK3 signaling

doi: 10.1038/ncomms7576

Figure Lengend Snippet: Sustained EphrinB2 phosphorylation in endothelial cells within the retrolental mass of PHPV. (a) Retrolental mass and retinal degeneration in PHPV. Cross section of PHPV eyeball stained with H&E (representative of 6 samples). Center panel: tiled image of the entire eye section showing the characteristic retrolental mass and retinal detachment; scale bar: 5mm. Left panel: magnified image showing retinal degeneration; scale bar: 100μm. Righteft panel: magnified image showing fibrovascular tissue within the retrolental mass; scale bar: 100μm. (b) Endothelial cells (CD31 + ) within the PHPV retrolental mass are p-EphrinB + and JNK3 − . Staining with anti-EphrinB (green), anti-JNK3 (white), anti-CD31 (red) and DAPI (blue). Arrowheads point to CD31 + p-EphrinB + JNK3 − cells. Scale bars: 1mm (left panel), 100μm (right panels) (c) Proximity co-localization of p-EphrinB and SHP2 in endothelial cells in PHPV retrolental mass. Red: PLA signal from p-EphrinB/SHP2 in section of PHPV retrolental mass; green: CD31 immunostaining; blue: DAPI. V: vessel. Arrowheads point to p-EphrinB + SHP2 + cells. Scale bars: 10μm. (d) PLA showing co-localization of EphrinB2 and STAT1 in endothelial cells from PHPV retrolental mass. Red: PLA signal from EphrinB2/STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrowheads point to EphrinB2 + STAT1 + cells. Scale bars: 50μm. (e) Co-localization of EphrinB2 and p-STAT1 is limited in endothelial cells in PHPV retrolental mass. Red: PLA signal from EphrinB2/p-STAT1, green: CD31; blue: DAPI. Asterisk: red blood cell. Arrows point toEphrinB2 + p-STAT1 + cells. Scale bars: 50μm.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit monoclonal or polyclonal IgG antibodies to: SHP2 (no. 3397; 1:1000), p-VEGFR2 (Tyr 1175 ) (no. 2478; 1:1000); VEGFR2 (no. 2479; 1:1000); p-Erk (Thr 202 /Tyr 204 ) (no. 4370; 1:2000); Erk (p44/p42) (no. 9102; 1:1000); p-EphrinB (Tyr 324/ Tyr 329 ) (no. 3481; 1:1000); cleaved PARP (Asp 214 ), (no. 5625; 1:1000); JNK3 (no. 2305; 1:1000); JAK2 (no. 3230; 1:1000), p-JAK2 (Tyr 1007/1008 ) (no. 3776; 1:1000); STAT1 (no. 9175; 1:1000), p-STAT1 (Tyr 701 ) (no. 9167; 1:1000), p-STAT3 (Tyr 705 ) (no. 9145; 1:2000) (all from Cell Signaling Technology); goat anti-SHP2 (Abcam no. Ab110194; 1:250); rabbit anti-JNK3 (Novus Biologicas no. NBP!-19542; 1:2000); goat anti-actin (Santa Cruz Biotechnology no. sc-1616; 1:).

Techniques: Staining, Immunostaining