Journal: Scientific Reports

Article Title: Coiled-coil domain containing 50-V2 protein positively regulates neurite outgrowth

doi: 10.1038/s41598-020-78304-3

Figure Lengend Snippet: An interaction between CCDC50-V2 and JNK in cells. ( A ) The interaction between CCDC50-V2 and JNK was detected in 293 T, A431, SH-SY5Y and HT22 cells by immunoprecipitation (IP) using anti-Flag-tagged beads, and the pellets were analysed by immunoblotting with the indicated antibodies. ( B ) The specific interaction between CCDC50-V2 and JNK2 was confirmed in 293 T cells co-transfected with JNK2 and CCDC50-V2 vectors by reverse IP using anti-Flag-tagged beads and an anti-JNK antibody with agarose beads. ( C ) CCDC50-V2 overexpression results in increased translocation of p–c-Jun from the cytosol to the nucleus. A431 and SH-SY5Y cells were transfected with the indicated vectors (0.5 μg/ml), harvested at 24 h, and separated into cytoplasmic and nuclear fractions. The blots were cropped from different parts of the same blot and full length blots are presented in Supplementary Figure . ( D ) To determine the JNK-specific binding region of CCDC50-V2, various deletion fragments of CCDC50-V2 were cloned into the pCMV6-entry vector (deletion constructs: 1–149 [D1], 150–324 [D2] and 325–482 [D3]). The protein–protein interactions between the V2-specific regions of CCDC50-V2 (D2) and JNK were investigated using deletion constructs in 293 T cells.

Article Snippet: Two-fold serial dilutions of purified GST-JNK1 and His-JNK2 proteins (Sino Biological Inc.) in binding buffer [20 mM HEPES (pH 7.4), 0.1 mM EDTA, 150 mM KCl, and 0.1% Triton-X100 (v/v)] were spotted onto a nitrocellulose membrane.

Techniques: Immunoprecipitation, Western Blot, Transfection, Over Expression, Translocation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Construct

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: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

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

Journal: Cell Death & Disease

Article Title: WWOX sensitises ovarian cancer cells to paclitaxel via modulation of the ER stress response

doi: 10.1038/cddis.2017.346

Figure Lengend Snippet: WWOX regulates ER stress through IRE-1 and PERK. Western blots showing the expression of WWOX and ER stress regulators IRE-1 (ab48187 and ab37073 antibodies; abcam), PERK (no. 3192; Cell Signalling Technology), JNK (MAB2076 (Total) and AF1205 (phospho) antibodies; R&D Systems) ( a ) untreated or treated with 8 nM paclitaxel for 24 h in PEO1 cell lines in the presence or absence of WWOX. ( b ) SKOV-3 and OVCAR-4 cells, 72 h after treatment with siWWOX or no targeting control and 24 h after treatment with paclitaxel in 50 nM SKOV-3 and 20 nM OVCAR-4 cells. β -Actin has been used as a loading control. Cell viability was measured through SRB assay in ( c ) WWOX-8 and Vector-9 ( d ) SKOV-3 (72 h siRNA treatment) and ( e ) OVCAR-4 cells (72 h siRNA treatment) with IRE-1 inhibitor (KIRA6; 532281; Calbiochem) and PERK (GSK2656157; S7033; Selleckchem) alone or together in combination with paclitaxel. Cells were treated with 1 μ g/μl inhibitors 2 h before paclitaxel treatment for 24 h. Triplicate experiments were performed

Article Snippet: The antibodies used in this study were as follows: rabbit anti-WWOX (a kind gift of Dr. M Aldaz, MD Anderson Cancer Centre, Houston, TX, USA) and ab137726 antibody (abcam), rabbit anti- β -actin (no. 4970; Cell Signalling Technology, Danvers, MA, USA), rabbit anti-Hsp60 (abcam; ab46798), rabbit anti-cleaved (Asp175) caspase-3 (R&D Systems, Minneapolis, MN, USA; 269518), mouse caspase-2 (no. 2224; Cell Signalling Technology), rabbit caspase-7 (no. 9492; Cell Signalling Technology), rabbit anti-phospho-eIF2A (Cell Signalling Technology; 9721), mouse anti-GRP78 (474421; R&D Systems), mouse anti-JNK1/JNK2 252323, MAB2076 and rabbit AF1205 (phospho-JNK) (R&D Systems), rabbit anti-phospho (T183/Y185)-JNK (AF1205), rabbit IRE-1 (ab48187 and ab37073; abcam) and PERK (no. 3192; Cell Signalling Technology).

Techniques: Western Blot, Expressing, Control, Sulforhodamine B Assay, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Activation of the stress-activated protein kinase JNK in response to herpes simplex virus-1 infection coordinates transition of BRD4 from chromosome association to transcription elongation

doi: 10.1016/j.jbc.2025.110590

Figure Lengend Snippet: HSV-1 infection requires JNK/MAPK pathway signaling. A , HSV-1 infection promotes phosphorylation of MAPK kinases. HeLa cells were infected with HSV-1 at an MOI of 0.3 for times as indicated. Protein phosphorylation and expression was detected by specific antibodies. The HSV-1 ICP0 and ICP4 were used as markers for infection. B , chemical inhibition of JNK on HSV-1 infection. Infected or uninfected control HeLa cells were mock-treated or treated with an MAPK inhibitor at 1 h prior to HSV-1 infection. The cells were harvested at 6 h post-infection and used for the detection of protein expression and phosphorylation. SP600125 (a JNK inhibitor, at 5 and 10 μM), SB203580 (a p38 inhibitor, at 0.3 and 1.0 μM), and U0126 (an ERK pathway inhibitor, at 1 and 3 μM). C and D , construction and confirmation of JNK knockout on HSV-1 infection. JNK ko HeLa cells were constructed and used for HSV-1 infection for 24 h. JNK ko and viral protein ICP4 production was determined by immunoblotting ( C ) and for infectious virus production ( D ). E , ectopic expression of JNK in JNK1/2 ko cells rescues HSV-1 infection. The JNK1/2 ko cells were transfected with plasmids encoding JNK1, JNK2, or both (JNK1+2) for 48 h, followed by HSV-1 infection another 36 h and then used for immunoblotting viral protein ICP0 expression. GAPDH was a loading control. The immunoblotting experiments were performed 3 times independently, while virus titers were from one representative experiment using triplicate samples. Data are mean ± SD, ∗, p ≤ 0.05, ∗∗∗, p ≤ 0.001.

Article Snippet: The plasmids for mammalian expression of JNK1 (HG10795-UT), JNK2 (HG10745-UT), and BRD4 were purchased from SinoBiological (Beijing), pcDNA5-CMV-FLAG-BRD4 wt was purchased from Miaoling ( P29409 , Wuhan), and pcDNA5-CMV-FLAG-BRD4-T1186A/T1212A was synthesized by GenScript.

Techniques: Infection, Phospho-proteomics, Expressing, Inhibition, Control, Knock-Out, Construct, Western Blot, Virus, Transfection

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A JNK1 and JNK2 protein expression in seven cultured human pancreatic cancer cell lines. Western Blot was performed using JNK (sc-571) antibody primarily detecting JNK1, but cross-reactive with JNK2 (anti-JNK1) and JNK (sc-572) antibody primarily detecting JNK2, but cross-reactive with JNK1 (anti-JNK-2). β-Actin served as a loading control (third panel). Total JNK activity was determined by assessing the amount of phosphorylated c-Jun (p–c-Jun, lower panel) in a nonradioactive in vitro kinase assay ( n = 1). Blots were scanned and relative JNK activity determined in relation to AsPC-1 cells. B , C Selective inhibition of JNK1 and JNK2 in PANC-1 ( B ) and MIA PaCa-2 ( C ). Cells were transfected in a stable manner with control shRNA (M-Neo-17/P-Neo-6), JNK1 shRNA (M-1–18, M-1–26/P-1–2, P-1–14), and JNK2 shRNA (M-2–12, M-2–24/P-2-2, P-2–5). The screening was performed using JNK antibodies (upper panels). β-Actin served as a loading control (third panel). C , D Total JNK activity was determined in MIA PaCa-2 by assessing the amount of phosphorylated c-Jun. A representative blot is shown in ( C ) and quantification of three independent experiments is shown in ( D ). JNK WT cells (WT and Neo clones), JNK1 KD clones, and JNK2 KD clones were analyzed together. ** p < 0.001 compared to JNK WT cells.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Expressing, Cell Culture, Western Blot, Control, Activity Assay, In Vitro, Kinase Assay, Inhibition, Transfection, shRNA, Clone Assay

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A , B Effect of the JNK inhibitor SP600125 on basal cell proliferation in WT cell lines ( A ) and after JNK KD ( B ). Indicated cells (10,000 cells/well) were cultured for 24 h in 96-well plates in complete medium followed by another 48 h in the absence or presence of increasing concentrations of SP600125 followed by the MTT assay. Results are shown as means (±SD) from three separate experiments with quadruplicate determinations compared to untreated control (DMSO only). B Cell proliferation was significantly inhibited at 10 µM SP600125 in wild-type (●) and Neo-17 (o). (C) Reduced JNK activity by SP600125 displayed by phosphorylated c-Jun. SP600125 markedly reduced JNK activity in MIA PaCa-2 and BxPC-3 cells while it was without effect in PANC-1. A representative blot is shown of three independent experiments. * p < 0.05 compared to JNK1 and JNK2 KD cells.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Cell Culture, MTT Assay, Control, Activity Assay

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: Resected pancreatic cancer specimens were immunohistochemically stained for JNK1 and 2 expressions (JNK1: ab110724, JNK2: ab76125). Examples for high expression are shown on the left, low expression on the right. Scale bar: 100 µm.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Staining, Expressing

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A , B Effect of JNK down-regulation on basal cell proliferation. Indicated cells (10,000 cells/well for MIA PaCa-2, 5000 cells/well for PANC-1) were cultured for 48 h in 96-well plates in complete medium and analyzed by the MTT assay. Results are shown as optical density and are means (±SD) from three separate experiments with quadruplicate determinations. C – F Anchorage-independent growth. MIA PaCa-2 was not influenced by JNK downregulation (C). JNK2 KD in PANC-1 increased colony formation and size while JNK1 KD showed opposing effects ( D , E ). Representative images of PANC-1 colony size (left, 2.5× magnification) and colony formation (right, scanned image after MTT staining) are shown in ( F ). Scale bar: 1 mm. Data are shown as means of three independent experiments of triplicate determinations ±SD. JNK WT cells (WT and Neo clones), JNK1 KD clones, and JNK2 KD clones were analyzed together. *** p < 0.001 compared to JNK WT cells.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Cell Culture, MTT Assay, Staining, Clone Assay

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A Single-cell movement. The total distance (μm in 24 h) of individual cells ( n = 30) covered within 24 h was evaluated using the ImageJ 1.32 and Simple Track program. Results are shown as the mean distance (±SD) in μm covered within 24 h. B , C In vitro invasion in the Boyden chamber assay. Indicated cells were seeded into the upper chamber and the number of cells that managed to invade the lower side of the chamber through 8 µm pores was determined after 36 h. B , C Migrated cells in % of JNK WT cells. Results are shown as mean (±SD) number of migrated cells in % of JNK WT cells within 36 h of three separate experiments. D Representative examples of the Boyden chamber assay. JNK WT cells (WT and Neo clones), JNK1 KD clones, and JNK2 KD clones were analyzed together. *** p < 0.001 compared to JNK WT cells.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: In Vitro, Boyden Chamber Assay, Clone Assay

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A Immunoblot analysis of N-Cadherin, E-Cadherin, Vimentin, and α-Tubulin in pancreatic cancer cell lines. β-Actin is used to confirm equal loading. B , C JNK2 downregulation leads to reduced Vimentin expression. MMP9 is upregulated in the KD of both isoforms, but predominantly in JNK1 KD while MMP2 expression is lost under JNK1 KD.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Western Blot, Expressing

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: A Tumor volume. JNK2 KD cells showed increased tumor volume, especially for M-2–12. B , C H.E. staining revealed solid pancreatic tumors in all xenografts. In control cells and JNK1 KD cells (M-1–18), the tumor was restricted to intrapancreatic growth while JNK2 KD clones regularly infiltrated surrounding organs. Tumors (TU) of M-2–12 (left) and M-2–24 (right) showed infiltration of the stomach (STO) and duodenum (DUO). The dashed line represents the tumor infiltrative line. This infiltrative pattern was not seen in control cells and JNK1 downregulated cells. Scale bar: 200 µm. * p < 0.05.

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Staining, Control, Clone Assay

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: Ki-67 staining corresponds to tumor volume with high staining in JNK2 KD clones. Ki-67 positivity score was counted in three sections per tumor and was displayed as means (±SD) of three sections: JNK WT: 8.2% pos., JNK1 KD: 6.6% pos., JKN2 KD: 53.5% pos. (first two lanes). Isoform KD was retained in vivo with reduced expression of either protein in the target clones compared to M-Neo-17. KD of either isoform reduced p–c-Jun levels in vivo (lanes 3–5).

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Staining, Clone Assay, In Vivo, Expressing

Journal: Cancer Gene Therapy

Article Title: c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1

doi: 10.1038/s41417-020-00290-5

Figure Lengend Snippet: Vimentin expression is strongly reduced after JNK2 KD but retained after JNK1 KD in vivo (first lane). MMP expression in JNK WT cells is generally low. MMP2 expression is upregulated after KD of both isoforms, but predominantly after JNK2 KD, while MMP9 expression is increased after JNK1 KD (lanes 2 and 3). Scale bar: 200 µm).

Article Snippet: Validated SureSilencing human JNK1, JNK2 shRNA, and control plasmids were obtained from SuperArray Bioscience Corp. (Frederick, MD).

Techniques: Expressing, In Vivo