Journal: Cellular Signalling

Article Title: Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

doi: 10.1016/j.cellsig.2018.09.021

Figure Lengend Snippet: EGF controls MSC proliferation via ILK and β-catenin activation. (A) MTT assay of viable cell numbers in two primary MSC donors following 24 and 48 h treatment with EGF and ILKi, graphs show mean absorbance ± SEM (n = 6), * = p < .05. (B) MTT assay of viable cell numbers in two primary MSC donors following 120 h treatment with EGF and increasing concentrations of an inhibitor of the AP-1 complex (AP-1i, concentrations = 17 nM, 34 nM and 68 nM), graphs show mean absorbance ± SEM (n = 6), * = p < .05, ** < 0.01 *** < 0.001. (C) EdU proliferation assay in two primary MSC donors following 48 h treatment with EGF, IWR-1 and Wnt3A, graphs show mean percentage EdU positive cells ± SEM ( n = 23), * = p < .05, ** < 0.01 *** < 0.001, **** < 0.0001. Donor 1 = K168, Donor 2 = K185. See also Fig. S4.

Article Snippet: Samples were treated with 10 nM EGF alone or in combination with the AP-1 inhibitor (17–68 nM, Biotechne, Cat. No. 2476) and were incubated for up to 120 h. Similarly, primary MSCs were treated with the Wnt pathway inhibitor (5 or 10 μM IWR-1, Sigma, Cat. No. I0161) with or without 10 nM EGF.

Techniques: Activation Assay, MTT Assay, Proliferation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Heme Oxygenase-1 Dysregulates Macrophage Polarization and the Immune Response to Helicobacter pylori

doi: 10.4049/jimmunol.1401075

Figure Lengend Snippet: Molecular regulation of hmox-1 transcription in macrophages. A, hmox-1 mRNA expression in RAW 264.7 cells pre-treated with ERKi, SP600125 (SP), SB203580 (SB), LY294002 (LY), Bay11-7082 (Bay), or SR11302 (SR) and infected with H. pylori for 6 h. ***P < 0.001 vs. Ctrl; §P < 0.05 vs. infected cells; n = 5. B, Western blots showing the effect of knockdown of p38 and p42/p44 in RAW 264.7 cells transfected with lmnA, p38, or erk1 siRNAs. C, Levels of hmox-1 mRNA in macrophages transfected with siRNAs directed against lmnA, p38, or erk1 and then treated with H. pylori for 6 h. ***P < 0.001, **P < 0.01 vs. Ctrl; §§P < 0.01 vs. cells transfected with lmnA or erk1 siRNA and infected with H. pylori; n = 5. D, Levels of p-p38 and p38 in macrophages pretreated with PP1 and infected with H. pylori or with the cagA mutant. Representative data of 3 independent experiments. E, p38 phosphorylation in RAW 264.7 cells infected with the strains 60190 or 7.13. F, Effect of nrf-2 siRNA on knockdown of NRF-2 in RAW 264.7 cells. G, Levels of hmox-1 mRNA in macrophages transfected with siRNAs directed against lmnA or nrf-2 and then treated with H. pylori for 6 h. ***P < 0.001 vs. Ctrl; §§§P < 0.001 vs. cells transfected with lmnA and infected with H. pylori; n = 6.

Article Snippet: The AP-1 inhibitor SR11302 (10 μM) was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Infection, Western Blot, Knockdown, Transfection, Mutagenesis, Phospho-proteomics

Journal: Experimental Biology and Medicine

Article Title: Highlight article: Telmisartan improves myocardial remodeling by inhibiting leptin autocrine activity and activating PPARγ

doi: 10.1177/1535370220908215

Figure Lengend Snippet: Ang II stimulates leptin synthesis and secretion in cardiac fibroblasts by promoting AP-1 nuclear translocation via the AT1R-ROS-ERK1/2 pathway. (a to c) Western blotting showing the expression of leptin in cardiac fibroblasts normalized to expression of β-actin in the same samples. Cardiac fibroblasts were pretreated with the AT1R antagonist telmisartan, the ROS scavenger NAC, or the ERK1/2 inhibitor PD98059 for 1 h or with siRNA for 24 h, before treatment with Ang II, the ROS-producing agent Dp44mT, or the ERK1/2 agonist C6 ceramide for 48 h. (d) Western blotting of AP-1 (c-JUN) by cardiac fibroblasts normalized to β-actin. Cardiac fibroblasts were pretreated with the ROS scavenger NAC, the ERK1/2 inhibitor PD98059, the p38 inhibitor SB205580, or the JNK inhibitor SP600125 for 1 h before treatment with Ang II for 48 h. (e) Western blotting of AP-1 (c-JUN) expression in the cytoplasm and nuclei of cardiac fibroblasts normalized to expression of β-actin or PCNA, respectively. Cardiac fibroblasts were pretreated with the ROS scavenger NAC or the ERK1/2 inhibitor PD98059 for 1 h, followed by treatment with Ang II for 48 h. (f) AP-1 transcriptional activity of cardiac fibroblasts evaluated by luciferase reporter assay. Cardiac fibroblasts pretreated with the ROS scavenger NAC, the ERK1/2 inhibitor PD98059, the p38 inhibitor SB205580, the JNK inhibitor SP600125, or the AP-1 inhibitor SR11302 for 1 h were incubated with Ang II for 48 h. (g) Level of leptin protein expression and mRNA transcription in cardiac fibroblasts, as determined by Western blotting and quantitative RT-PCR, respectively. Cardiac fibroblasts were incubated in the presence or absence of the AP-1 inhibitor SR11302 for 1 h before treatment with Ang II for 48 h. Ang II: angiotensin II; AP-1: activation protein 1. N = 4–6. Data are reported as mean ± SEM; *P < 0.05 vs. control group; #P < 0.05 vs. Ang II, Dp44mT or C6 ceramide group.

Article Snippet: The cells were pretreated for 1 h with the leptin antagonist Leptin tA (PROSPEC, Israel), the AT1R antagonist telmisartan, the ROS scavenger NAC, the ROS-producing agent Dp44mT, pioglitazone (Sigma, St. Louis, MO, USA), the ERK1/2 inhibitor PD98059, the ERK1/2 agonist C6 ceramide, the p38 inhibitor SB205580, the JNK inhibitor SP600125, the AP-1 inhibitor SR11302, the JAK2 inhibitor AG-490, the STAT3 inhibitor S3I-201, or the PPAR-γ inhibitor GW9662 (Selleck Chemicals LLC, USA) prior to treatment with Ang II (Sigma) or leptin (PeproTech, Rocky, USA) for 48 h.

Techniques: Translocation Assay, Western Blot, Expressing, Activity Assay, Luciferase, Reporter Assay, Incubation, Quantitative RT-PCR, Activation Assay, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Melanoma-derived versican reactivates tumor-associated macrophages by upregulating pyruvate carboxylase through TLR2-MyD88-RelB axis under normoxia

doi: 10.3724/abbs.2025011

Figure Lengend Snippet: TCM upregulates PCB in TAMs through the MyD88-NF-κB pathway (A) Representative western blots of PCB in BMDMs treated with control medium, TCM, LPS (50 ng/mL), IFNγ (20 ng/mL), or IL4 (50 ng/mL) for 36–48 h. (B) The mRNA levels of PCB in BMDMs treated with control medium, TCM, LPS (50 ng/mL), IFNγ (20 ng/mL) or IL4 (50 ng/mL) for 36–48 h. (C) BMDMs isolated from WT or MyD88–/– mice were treated with LPS or TCM for 24 or 48 h, and the expression of PCB was analyzed by western blot analysis. (D) Representative western blots of PCB in BMDMs treated with LPS (50 ng/mL) alone or with LPS combined with the NF-κB inhibitor BAY11-7082 (1 μM). (E) Representative western blots of PCB in BMDMs treated with TCM alone or with TCM in combination with BAY11-7082 at the indicated concentrations. (F) Representative western blots of PCB in BMDMs treated with TCM (upper panel) or LPS (lower panel) in the presence of vehicle or different doses of the AP-1 inhibitor SR113021. (G) The mRNA levels of PCB in BMDMs treated as indicated. (H) BMDMs isolated from WT or MyD88–/– mice were treated with different doses of TCM or TNFα, and the expression of PCB was analyzed by western blotting 48 h later. **P < 0.01, ***P < 0.001 by Student’s t test and one-way ANOVA. ns, not significant.

Article Snippet: BAY 11-7082 (HY-13453), SR113021 (HY-15870) and C29 (HY-100461) were purchased from MCE (Monmouth Junction, USA).

Techniques: Western Blot, Control, Isolation, Expressing

Journal: Investigative ophthalmology & visual science

Article Title: Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

doi: 10.1167/iovs.64.12.27

Figure Lengend Snippet: FIGURE 7. JunB binds to and regulates Netrin-1 and Slit2 in Aqp5−/−mice LG epithelial cells. (A) Heatmap analysis of mRNA profiles about transcription factors that co-transcribe Netrin-1 and Slit2 in LG of Aqp5+/+ and Aqp5−/−. (B) The qRT-PCR analysis of the expression of JunB, Jun, JunD, and Fosl in LG tissues (n = 4; mean ± SD; n.s., not significant; *** P < 0.001; Student’s t-test). (C) Western blotting analysis showed the expression level JunB in LG epithelial cells. (D) ChIP-PCR analyses of JunB enrichment at binding sites in the murine Netrin-1 and Slit2 promoter. (E) The qRT-PCR analysis of the expression of Netrin-1 and Slit2 in Aqp5+/+ and Aqp5−/−LG epithelial cells treated with SR11302 or DMSO 24 hours (n = 4; mean ± SD; * P < 0.05; *** P < 0.001; Student’s t-test and Mann-Whitney test). (F) Western blot analysis of content of protein of Netrin-1 and Slit2 in Aqp5+/+ and Aqp5−/−LG epithelial cells treated with SR11302 or DMSO 24 hours. (G) Densitometric analyses of Western blots presented in (F) (n = 3; mean ± SD; * P < 0.05; ** P < 0.01; Student’s t-test)

Article Snippet: SR11302 (#T23384, TargetMol; TOPSCIENCE, Shanghai, China) was added to the cells at 10 μM, and an equal volume of DMSO was added to the control group.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Epicardial Adipose Tissue–Derived Leptin Promotes Myocardial Injury in Metabolic Syndrome Rats Through PKC / NADPH Oxidase/ ROS Pathway

doi: 10.1161/JAHA.123.029415

Figure Lengend Snippet: H9C2 cardiomyocytes were pretreated with the PKC inhibitor GF, the NADPH oxidase inhibitor Apo, or the ROS scavenger NAC for 1 hour before treatment with leptin (100 ng/mL) for 48 hours. A , Concentrations of IL‐6 and TNF‐α in the supernatants of H9C2 cardiomyocytes measured by enzyme‐linked immunosorbent assay. B , Western blotting of AP‐1 (c‐JUN) by H9C2 cardiomyocytes normalized to β‐actin. C , Western blotting of AP‐1 (c‐JUN) expression in the cytoplasm and nuclei of H9C2 cardiomyocytes normalized to expression of β‐actin or proliferating cell nuclear antigen, respectively. H9C2 cardiomyocytes were pretreated with the NADPH oxidase inhibitor Apo or ROS scavenger NAC for 1 hour before treatment with leptin (100 ng/mL) for 48 hours. D , AP‐1 transcriptional activity of H9C2 cardiomyocytes evaluated by luciferase reporter assay. H9C2 cardiomyocytes were pretreated with the PKC inhibitor GF, the NADPH oxidase inhibitor Apo, the ROS scavenger NAC, or the AP‐1 inhibitor SR for 1 hour before treatment with leptin (100 ng/mL) for 48 hours. E , Concentrations of IL‐6 and TNF‐α in the supernatants of H9C2 cardiomyocytes, as determined by enzyme‐linked immunosorbent assay. H9C2 cardiomyocytes were pretreated with the AP‐1 inhibitor SR for 1 hour before treatment with leptin (100 ng/mL) for 48 hours. For A , D, and E , n=6 per group; for B and C , n=4 per group. Data are reported as mean±SEM. * P <0.05 vs the control group; ** P <0.01 vs the control group; *** P <0.001 vs the control group; # P <0.05 vs the leptin group; ## P <0.01 vs the leptin group; ### P <0.001 vs the leptin group. AP‐1 indicates activation protein 1; Apo, apocynin; Con, control rats; GF, GF109203X; IL‐6, interleukin 6; NAC, N‐acetyl‐L‐cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; PKC, protein kinase C; ROS, reactive oxygen species; SR, SR11302; and TNF‐α, tumor necrosis factor α.

Article Snippet: The H9C2 rat cardiomyoblasts were pretreated for 1 hour with the leptin antagonist Leptin tA (PROSPEC), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (Selleck Chemicals LLC), the protein kinase C (PKC) inhibitor GF109203X (Selleck Chemicals LLC), the reactive oxygen species (ROS) scavenger N‐acetyl‐L‐cysteine (NAC; Sigma Chemical), the activator protein 1 (AP‐1) inhibitor SR11302 (Selleck Chemicals LLC), the mitochondrial‐targeted antioxidant MitoQ (Cayman Chemical), or the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (Selleck Chemicals LLC), before the treatment with leptin (PeproTech), control rat EAT‐CM, or MetS rat EAT‐CM for 48 hours.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay, Luciferase, Reporter Assay, Control, Activation Assay

Journal: The Journal of Reproduction and Development

Article Title: Advanced glycation end products regulate interleukin-1β production in human placenta

doi: 10.1262/jrd.2017-032

Figure Lengend Snippet: Intracellular AGE signaling pathways on inflammatory cytokine production in the Sw.71 human placental cell line. Sw.71 cells were pre-incubated for 1 h with specific pharmacological inhibitors (SB203580 as a p38 MAPK inhibitor, SP600125 as a JNK inhibitor, Bay11-7082 as a NF-κB inhibitor, and SR11302 as an AP-1 inhibitor). (A) After inhibitor treatments, cells were incubated for 24 h with AGEs (200 μg/ml). (B) After inhibitor treatments, cells were incubated for 24 h with AGEs (200 μg/ml), followed by nano-silica (100 μg/ml) treatment for 24 h. (C) After inhibitor treatments, cells were incubated for 24 h with AGEs (200 μg/ml). Intracellular IL-1β, and IL-1β and IL-6 levels in the supernatant were measured using ELISA. Data are expressed as mean ± SEM (n = 4). Significant differences were identified using ANOVA, followed by Bonferroni’s multiple comparison test; * P < 0.05.

Article Snippet: To further explore the relationships between AGEs and inflammatory cytokine production, various pharmacological inhibitors, including SB203580 [a mitogen-activated protein kinase (MAPK) p38 inhibitor, 20 μM, Merck Millipore, Belize, MA, USA], SP600125 [a Jun N-terminal kinase (JNK) inhibitor, 20 μM, Merck Millipore], Bay 11-7082 (a NF-κB activation inhibitor, 2 μM, Merck Millipore), SR11302 (an AP-1 inhibitor, 10 μM, Merck Millipore), N -acetyl-L-cysteine (NAC, an antioxidant, 5 mM, Wako Pure Chemical Industries), or diphenyleneiodonium (DPI, a NADPH inhibitor, 2 μM, Sigma-Aldrich) were pre-incubated in the medium for 1 h. Supernatant and cell lysate were collected for ELISA, real-time RT-PCR, or western blot analysis, and stored at –20°C or –80°C before use.

Techniques: Protein-Protein interactions, Incubation, Enzyme-linked Immunosorbent Assay, Comparison