The FJU‐Cs compound used in this study was synthesized at the Department of Chemistry at Fu‐Jen Catholic University, Taiwan. LPS (Escherichia coli 0111:B4; Catalogue Number: L4391) was purchased from Sigma‐Aldrich. BAY11‐7082 (NF‐κB inhibitor), PD98059 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) were purchased from Enzo Life Sciences. Rapamycin (mTOR inhibitor) and Wortmannin (Phosphatidylinositol 3‐kinase Inhibitor) was purchased from Abcam Biotechnology. CLI‐095 (TLR4 signaling inhibitor) was purchased from InvivoGen.
Sp600125
Manufactured by Enzo Life Sciences
Sourced in United States, Germany
SP600125 is a potent, selective, and cell-permeable inhibitor of the c-Jun N-terminal kinase (JNK) pathway. It inhibits the phosphorylation of c-Jun and other JNK substrates, making it a useful tool for studying the role of the JNK signaling pathway in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by Enzo Life Sciences (18 mentions)
U0126, by Enzo Life Sciences (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Pd98059, by Enzo Life Sciences (10 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Bay11 7082, by Enzo Life Sciences (5 mentions)
Sb202190, by Enzo Life Sciences (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Z vad fmk, by R&D Systems (3 mentions)
Actinomycin d, by Merck Group (3 mentions)
Anti phospho jnk, by Cell Signaling Technology (3 mentions)
Hybond c membrane, by GE Healthcare (3 mentions)
Ag1478, by Enzo Life Sciences (3 mentions)
Ly294002, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Welgene (2 mentions)
Mtt assay, by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Anti zo 1 cat, by Santa Cruz Biotechnology (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
46 protocols using sp600125
1
Inhibitors of Inflammatory Pathways
2
Inhibition of Signaling Pathways in RCC
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Inhibitors for AP-1 (NDGA), NF-κB (PDTC), NADPH oxidase (Nox) (DPI) and the antioxidant N-acetyl l -cysteine (NAC) were from Sigma. Inhibitors for JNK (SP600125), p38 (SB202190) and MEK/ERK (U0126) were from Enzo. Inhibitors for caspase-8 (z-IETD-FMK), -9 (z-LEHD-FMK), -10 (z-AEVD-FMK) and pan-caspase inhibitor (z-VAD-FMK) were from R&D Systems (supplied by Bio-Techne, Abingdon, UK). NAC was reconstituted in DR/5% medium and its pH adjusted to 7.0 using 1 M NaOH solution, and was filter-sterilised before use. All other inhibitors were reconstituted in DMSO (Sigma). RCC cells were co-cultured with 3T3Neo or 3T3CD40L in the presence of inhibitors for 48 h and apoptosis determined as above. Cells treated with DMSO alone (vehicle controls) were included.
3
Cytotoxic Effects of Pinosylvin on Leukemia Cells
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Human monocytic cell lines THP-1 and U937 cells originated from acute monocytic leukemia and myelomonocytic lymphoma, respectively. THP-1 and U937 cells were cultured in Roswell Park Memorial Institute Medium 1640 (RPMI 1640, Welgene, Seoul, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene), 2 mmol/L L-glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin. THP-1 and U937 cells were grown in a humidified incubator with 5% CO2 atmosphere at 37°C. These cells were then starved for 12 h in starvation media (RPMI 1640 supplemented with 50 μg/ml penicillin and 50 μg/ml streptomycin). Various concentrations (0–100 μmol/L) of pinosylvin were used to obtain a dose-curve, while 0.1 μmol/L of pinosylvin was used for a time-course experiment. We also treated cells with different inhibitors when necessary. Those inhibitors included PD146176 (an ALOX15 inhibitor; 10 μmol/L; Enzo life sciences, New York city, NY, USA), PD98059 (an ERK inhibitor; 50 μmol/L, Enzo life sciences) and SP600125 (a JNK inhibitor; 50 μmol/L, Enzo life sciences). They were utilized for pretreatment for 1 h before treatment with pinosylvin.
4
Antibody Characterization for Signaling Pathways
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SP600125 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Antibodies against phospho-ERK 1/2 (Thr-202/Tyr204), phospho-p38 MAPK (Thr-180/Tyr-182), phospho-JNK 1/2 (Thr183/Tyr185) and phospho-EGFR (Tyr1068) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal antibody against β-actin was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Antibody against γ-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MUC5AC antibody was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibody against HO-1 was purchased from Abcam (UK).
5
Lens Proteomics and Microscopy
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E13 lenses were placed in Complete Medium (Medium 199 [Life Technologies, 11150-059] with 10% fetal bovine serum, 1% penicillin and 1% streptomycin) at 37 °C. Lenses free of opacities after 2 h in culture were treated for 24 h or 48 h, as indicated, with either the specific MAPK/JNK inhibitor SP600125 (25 µM; Enzo Life Sciences, BML-EI305-0010),66 (link) the specific and irreversible MAPK/JNK inhibitor JNK-IN-867 (link) (1 μM; EMD Millipore, 420150), the MTOR kinase inhibitor/autophagy inducer, rapamycin (100 nM; Calbiochem/EMD Millipore, 553211), or the vehicle control DMSO (0.1% Me2SO; Sigma, 276855). Lenses were microdissected for immunoprecipitation or direct western blot analysis, fixed, and cryosectioned for immunostaining studies, or fixed and prepared for electron microscopic studies.
6
Apoptosis Regulation Pathway Analysis
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Anti-Bcl-xL (sc-634, 1:700), anti-p53 (sc-126, 1:1000), anti-p21 (sc-6246, 1:700), anti-Mcl-1 (sc-819, 1:1000), and anti-c-FLIP (sc-8347, 1:700) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-β-actin (A5441, 1:2000) antibody and glutathione ethyl ester (GEE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti- poly (ADP-ribose) polymerase (PARP) (#9542, 1:1000) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-caspase-3 (610322, 1:1000) antibody was purchased from BD Biosciences (Bedford, MA, USA). Benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was purchased from R&D Systems (Minneapolis, MN, USA). PD-98059 (a MEK inhibitor; PD), SP600125 (a JNK inhibitor; SP), and SB-203580 (a p38 MAP Kinase inhibitor; SB) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). N-acetylcysteine (NAC) was purchased from Calbiochem (San Diego, CA, USA).
7
Fisetin Modulates Inflammatory Markers in BEAS-2B Cells
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BEAS-2B cells (2 × 105 cells/mL) were treated with 0–30 μM fisetin at 37 °C for 1 h and then incubated with 10 ng/mL TNF-α or 10 ng/mL TNF-α/IL-4 at 37 °C for 24 h. The supernatants were collected to detect the levels of intercellular adhesion molecule-1 (ICAM-1), IL-8, IL-6, MCP-1, and c-c motif chemokine ligand CCL5, CCL11, and CCL24 using sandwich ELISA kits (R&D, Minneapolis, MN, USA). Specific protein levels were detected using a microplate reader (Thermo) at an OD of 450 nm as described previously [25 (link)]. Fisetin (10 μM) was combined with MAPK specific inhibitors, including 10 μM SP600125 (JNK inhibitor), 10 μM SB203580 (p38 inhibitor), and 10 μM PD98059 (ERK inhibitor) (Enzo Life Sciences, Inc., Farmingdale, NY, USA), to detect the ICAM-1 protein level by ELISA.
8
Evaluation of Apoptosis Inducers
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Sodium azide, anisomycin and doxycycline (dox) were purchased from Sigma (St Louis, MO, USA). zVAD-fmk, SP600125 and staurosporine were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Etoposide was purchased from EBEWE Pharma (Kundl, Austria) and cisplatin was purchased from TEVA ABIC (Netanya, Israel).
9
Apoptosis Induction in Hepatocytes
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Hepatocytes were isolated and cultured as previously described [56 (link)]. After 24 h, cells were treated with the gene transcription inhibitor actinomycin D (ActD, 200 ng/mL) in combination with murine TNF (10 ng/mL, both from Sigma-Aldrich, Hamburg, Germany) or Jo2 (50 ng/mL, BD Pharmingen), to induce death receptor-mediated apoptosis. Where indicated the pan caspase inhibitor zVAD (50 µM), the JNK inhibitor SP600125 (100 µM, both from Enzo Life Sciences, Lörrach, Germany), or the IKK inhibitor BAY-11-7082 (10 µM, Calbiochem, EMD Chemicals, Inc., San Diego, CA, USA) was added 1 h prior to ActD/TNF or Jo2 treatment. Cell survival was assessed by MTT assay (Sigma-Aldrich) at the indicated time points.
10
Oxidative Stress Modulation Protocols
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The AP-1 inhibitor (NDGA), NADPH oxidase inhibitor (DPI), Thioredoxin inhibitor (PX-12), and the antioxidants N-acetyl L-cysteine (NAC) and propyl gallate were obtained from Sigma. The JNK inhibitor (SP600125) was obtained from Enzo. NAC was initially reconstituted in DR 5% culture medium at 30 mM , its pH was adjusted to 7.0 using 1M NaOH solution and was filter-sterilised before use. Other inhibitors were reconstituted in DMSO (Sigma) or ethanol (propyl gallate) and appropriate solvent-only controls were included in all experiments. For some experiments, cells were treated with hydrogen peroxide (H2O2) (Sigma) diluted in culture medium at the indicated concentrations for 48 h. The effects of H2O2 on cell growth were quantified by measuring cell biomass using the CellTiter 96 AQueous One cell proliferation assay (Promega) and results were expressed as % cell growth of each condition in comparison to untreated controls, as detailed elsewhere.63 (link)
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