Journal: Journal of Clinical Investigation

Article Title: Collagen VII plays a dual role in wound healing

doi: 10.1172/jci68127

Figure Lengend Snippet: Figure 5 Mechanisms of delayed wound closure in Col7a1-hypomorphic keratinocytes. (A) In in vitro assays, wound closure by keratinocytes was assessed at 8, 20, and 24 hours. At 20 and 24 hours, Col7a1-hypomorphic keratinocytes closed wounds at a reduced rate. n = 6; values rep- resent mean ± SEM. *P < 0.05. (B–D) Western blotting of wild-type and Col7a1-hypomorphic keratinocyte protein lysates revealed increased expression of integrin β4 subunit (B) and increased phosphorylation of Ser473 AKT (C) and JNK2 (D) in cultured Col7a1-hypomorphic keratino- cytes. Densitometric quantification of keratinocyte isolations is also shown. n ≥ 3; values represent mean ± SD. *P < 0.05; **P < 0.01. (E) Western blots on cell lysates from tamoxifen-induced Col7a1 knockout mouse keratinocytes. Keratinocytes from the same isolation were divided into 2 pools: one was treated with 4-OH tamoxifen to induce Col7a1 knockout, the other received only DMSO and served as control. The changes in Col7a1-hypomorphic keratinocytes were replicated in keratinocytes after forced COL7A1 loss. Loss of COL7A1 did not affect expression of lam- inin-332 (LM-β3/γ2; representing the laminin β3 and γ2 chains of laminin-332), but increased expression of integrin β4 and phosphorylation of Ser473 AKT and JNK2. Total AKT and GAPDH were used to ensure equal loading. (F) Altered Laminin-332 organization in Col7a1-hypomorphic keratinocytes. Primary keratinocytes of the indicated genotypes and treatments were cultured for 24 hours in the presence of ascorbate and stained for COL7A1 (red) and laminin-332 (green). Original magnification, ×400. Tam, tamoxifen.

Article Snippet: The following antibodies were used: mouse antiGAPDH (Millipore), rabbit anti–COL7A1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), rabbit anti-tubulin (Abcam), rat anti–integrin α6 (GoH3) (Progen), rat anti–integrin β1 (BD Biosciences), rat anti–integrin β4 (BD Biosciences), rabbit anti–integrin β4 (Santa Cruz Biotechnology), mouse anti–active JNK1 and JNK2, rabbit anti-JNK (Abcam), rabbit anti–phospho–Tyr705 STAT3 (Cell Signaling Technology), mouse anti-AKT (Santa Cruz Biotechnology), rabbit anti–phospho–Ser473 AKT (Cell Signaling Technology), rat anti–Ki-67 (Dako), Cy3-conjugated mouse anti–α-SMA (Sigma-Aldrich), rat anti-CD11b (BD Biosciences), rabbit anti-mouse collagen I (Acris), rabbit anti–laminin-332 (gift from K. Yancey, University of Texas Southwestern, Dallas, Texas, USA), rabbit anti–pan-laminin (Progen), and rat anti–laminin α5 (gift from L. Sorokin, Muenster University, Muenster, Germany).

Techniques: In Vitro, Western Blot, Expressing, Phospho-proteomics, Cell Culture, Knock-Out, Isolation, Control, Staining

Journal: Oncotarget

Article Title: SETD2 loss sensitizes cells to PI3Kβ and AKT inhibition

doi: 10.18632/oncotarget.26567

Figure Lengend Snippet: ( A ) Bar graph showing relative cell viability as a percentage of control (si-CTL) of SETD2 (+/+) 786-0 and SETD2 (KO) 786-0 and (–/–) A498 cells in response to treatment with siRNA specific to p110α, p110β, and p110δ (singlets and doublets). * P < 0.05; ** P < 0.005; *** P < 0.001; **** P < 0.0001; ns, no statistical significance observed. Standard deviations were calculated and represented for all conditions. ( B ) Western blot analysis of indicated proteins showing decrease in expression of p110α, p110β, and p110δ in individual or combinatorial treatments (specificity) and variations in phosphorylation levels of known downstream target pAKT-S473 (efficacy). Actin is a loading control.

Article Snippet: Cells were transfected with human SMART pool ON-TARGETplus PIK3CB (L-003019-00-0005), PIK3CA (L-003018-00-0005), PIK3CD (L-006775-00-0005), and ON-TARGETplus Non-targeting pool (D-001810-10-05) siRNAs using DharmaFECT 1 transfection reagent (T-2-001-02) (GE Dharmacon) or with human PI3-Kinase p110α (sc-39127), PI3-Kinase p110β (sc-37269), PI3-Kinase p110δ (sc-39101), and Control siRNA-A (sc37007) siRNAs (Santa Cruz Biotechnology) using Lipofectamine RNAiMax Transfection Reagent (Invitrogen) according to the manufacturer's protocol.

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

Journal: Antioxidants

Article Title: Oxidative Stress Triggers Porcine Ovarian Granulosa Cell Apoptosis Through MAPK Signaling

doi: 10.3390/antiox14080978

Figure Lengend Snippet: MAPK signaling pathway is significantly upregulated in porcine AF. ( A – H ) RT-qPCR analysis of mRNA expression levels of ASK1, TRADD, JUN, MKK7, MKK4, JNK2, JNK3, and TP53 in porcine HF and AF. ( I ) Healthy follicles of 3–5 mm were randomly assigned to the control group, H 2 O 2 treatment group, and selonsertib pretreatment group. ( J–P ) The expression levels of ASK1, TRADD, JUN, MKK4, JNK2, JNK3, and TP53 in the control group, H 2 O 2 treatment group, and selonsertib pretreatment group were detected by RT-qPCR. Data are presented as the mean ± SEM from a minimum of three independent biological replicates. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Antibodies against Phospho-JNK1-T183/Y185 + JNK2-T183/Y185 + JNK3-T221/Y223 Rabbit mAb (AP1451), JNK2 Rabbit pAb (A1251), TRADD Rabbit pAb (A18626), MAP2K4 Rabbit pAb (A21597), and Phospho-MAP2K4-S257/T261 Rabbit pAb (AP0541) were purchased from ABclonal.

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Antioxidants

Article Title: Oxidative Stress Triggers Porcine Ovarian Granulosa Cell Apoptosis Through MAPK Signaling

doi: 10.3390/antiox14080978

Figure Lengend Snippet: ASK1 inhibition protects mouse GCs from apoptotic cell death. ( A ) CCK-8 was used to detect the cell viability of the control group, H 2 O 2 treatment group, and selonsertib pretreatment group. ( B – K ) The expression levels of ASK1, TRADD, JUN, MKK4, JNK2, JNK3, TP53, CASP3, and BAX in the control group, H 2 O 2 treatment group, and selonsertib pretreatment group were detected by RT-qPCR. Data represented the mean ± SEM of at least three biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: Antibodies against Phospho-JNK1-T183/Y185 + JNK2-T183/Y185 + JNK3-T221/Y223 Rabbit mAb (AP1451), JNK2 Rabbit pAb (A1251), TRADD Rabbit pAb (A18626), MAP2K4 Rabbit pAb (A21597), and Phospho-MAP2K4-S257/T261 Rabbit pAb (AP0541) were purchased from ABclonal.

Techniques: Inhibition, CCK-8 Assay, Control, Expressing, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Oxidative Stress Triggers Porcine Ovarian Granulosa Cell Apoptosis Through MAPK Signaling

doi: 10.3390/antiox14080978

Figure Lengend Snippet: Inhibition of ASK1 rescued the apoptosis of GC. ( A – G ) Western blot was used to detect the expression levels of P-ASK1, TRADD, P-MKK4, JNK2, P-JNK1/2/3, and CL-CASP3 in the control group, H 2 O 2 treatment group, and selonsertib pretreatment group. ( H ) ROS staining is used to detect reactive oxygen species in the control group, H 2 O 2 treatment group, and selonsertib pretreatment group. ( I ) Mito Tracker staining is used to detect mitochondrial activity in the control group, H 2 O 2 treatment group, and selonsertib pretreatment group. Scale bars in ( H , I ) are 100 µm. Data are presented as the mean ± SEM from a minimum of three independent biological replicates. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Antibodies against Phospho-JNK1-T183/Y185 + JNK2-T183/Y185 + JNK3-T221/Y223 Rabbit mAb (AP1451), JNK2 Rabbit pAb (A1251), TRADD Rabbit pAb (A18626), MAP2K4 Rabbit pAb (A21597), and Phospho-MAP2K4-S257/T261 Rabbit pAb (AP0541) were purchased from ABclonal.

Techniques: Inhibition, Western Blot, Expressing, Control, Staining, 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 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: Cancer Research

Article Title: JNK Signaling Mediates EPHA2-Dependent Tumor Cell Proliferation, Motility, and Cancer Stem Cell–like Properties in Non–Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-13-2136

Figure Lengend Snippet: Figure 3. JNK/c-JUN signaling mediates EPHA2-dependent tumor cell growth and motility. A and B, A549 or H1975 cells were treated with DMSO or 10 mmol/L JNK inhibitor SP600125 for 24 hours. Cell growth (A) and motility (B) in response to 10% serum in the presence of DMSO or SP600125 were assessed by colony formation assay and Transwell migration assay, respectively. Shown are images from one experiment that are representative of three independent experiments. Data were pooled and presented as mean SEM. C, EPHA2 knockdown A549 or H1975 cells were transduced with retroviruses expressing wild-type EPHA2, constitutively activated JNK1-CA, JNK2-CA, wild-type c-JUN, or vector control. Expression of JNK1-CA, JNK2-CA, or c-JUN, as well as phosphorylation of JNK and c-JUN was analyzed by Western blot analysis. Densitometry analyses for three independent experiments for each condition are summarized in the bar graph. D and E, the effects of JNK1-CA or JNK2- CA on clonogenic cell growth (D) and motility (E) were determined by colony formation assay and Transwell migration assay, respectively. Shown are images from one experiment that are representative of three independent experiments. , P < 0.05; , P < 0.01 (two-tailed Student t test).

Article Snippet: Human c-JUN cDNA and constitutively activated c-JUN-NH2-kinase (JNK)-1 and JNK2 were obtained from Addgene and subcloned into pCLXSN retroviral vector.

Techniques: Colony Assay, Transwell Migration Assay, Knockdown, Transduction, Expressing, Plasmid Preparation, Control, Phospho-proteomics, Western Blot, Two Tailed Test

Journal: JOR Spine

Article Title: Intracellular Detection of C. acnes and S. aureus in Non‐Herniated Human Intervertebral Discs: Implications for Catabolic Signaling Pathways

doi: 10.1002/jsp2.70139

Figure Lengend Snippet: Stimulation of NP cells with S. aureus and C. acnes combined with inhibitors. In cell‐ELISA was used to measure phospho‐ ERK, JNK, p38 and NFkB. Cells in monolayer under hypoxia were left to attach overnight before treatment with the inhibitors for TLR2, NODin1 and both combined for 24 h. Followed by the inhibition was treatment with 50 μg/mL PGN, S. aureus (MOI 0.01) and C. acnes (MOI 0.01) for 2 h in combination with the inhibitors. The different shades of blue represent the different donors ( N = 3 donors, n = 3 wells, (stats performed on n = 9)). All the measured concentrations were normalized to the non‐treated control. The No treatment graphs, show the different levels of phospho ERK, JNK, p38 and NFkB for cells only inhibited but not treated with a stimulant. No Inhibition shows treatment with PGN, S. aureus and C. acnes without inhibitors and TLR2, NODin1 and Combined Inhibition show the treatment combined with the inhibition. Statistical analysis was performed using Kruskal Wallis multiple comparison against the control group of each treatment ( p = 0.05).

Article Snippet: Protein phosphorylation was measured for c‐Jun‐N‐terminal kinase (JNK, CBEL‐JNK‐1, RayBiotech, GA 30092, US) p38 mitogen‐activated protein kinase (p38, CBEL‐P38, RayBiotech), extracellular signal‐regulated kinase (ERK, CBEL‐ERK, RayBiotech) and nuclear factor kappa B p65 (NFkB, FBCAB0073, Assay Genie, Dublin Ireland) as per the manufacturer's instructions.

Techniques: In-Cell ELISA, Inhibition, Control, Comparison