Acute allergic airway inflammation was induced according to our previous study and modified.30 (link) On Day 0 and 7, mice were sensitized with 0.5 mL suspension consisting of 10 μg OVA (Sigma) and 1 mg KAl(SO4)2 (Sangon Biotech, Shanghai, China) in saline intraperitoneally. Then from Day 14 to 20, mice were challenged with 1% OVA by airway inhalation 30 min every day. The mice were randomly grouped into two parts. Part 1 comprised four groups (control, CpG-ODN, OVA, OVA + CpG-ODN), n = 6 for each; Part 2 comprised four groups (OVA, OVA + SP600125, OVA + CpG-ODN, OVA + CpG-ODN + SP600125), n = 6 for each. For CpG-ODN, OVA + CpG-ODN and OVA + CpG-ODN + SP600125 groups, 30 μg of CpG-ODN were given intraperitoneally to mice one hour before OVA challenge. For OVA + SP600125, OVA + CpG-ODN + SP600125 groups, SP600125 (30 mg/kg, Sigma) were given intraperitoneally to mice two hours before OVA challenge.
Sp600125
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Macao, China, Sao Tome and Principe, Morocco, Sweden, Japan, Switzerland, France, India, Italy, Canada, Brazil
SP600125 is a small molecule compound that functions as a selective inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway. It is commonly used as a research tool to investigate the role of the JNK pathway in various cellular processes and disease models.
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Lab products found in correlation
Sb203580, by Merck Group (221 mentions)
Pd98059, by Merck Group (138 mentions)
U0126, by Merck Group (81 mentions)
Ly294002, by Merck Group (77 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (67 mentions)
Sb202190, by Merck Group (47 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (38 mentions)
Bay11 7082, by Merck Group (37 mentions)
β actin, by Cell Signaling Technology (20 mentions)
U0126, by Cell Signaling Technology (20 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (19 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (17 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (17 mentions)
β actin, by Santa Cruz Biotechnology (16 mentions)
Wortmannin, by Merck Group (15 mentions)
P jnk, by Cell Signaling Technology (14 mentions)
Cleaved caspase 3, by Cell Signaling Technology (14 mentions)
Penicillin, by Thermo Fisher Scientific (14 mentions)
Lipopolysaccharides (lps), by Merck Group (13 mentions)
643 protocols using sp600125
1
Allergic Airway Inflammation Model with CpG-ODN and SP600125
2
Signaling Pathways in Cell Stress Response
3
Murine Melanoma Cell Inhibition Assay
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B16F10 murine melanoma cells were routinely maintained in Dulbecco's.
Modified Eagle Medium (DMEM) with 1.5 g/L sodium bicarbonate, 25 mM glucose, and 10% fetal bovine serum (FBS), as previously described 14 . EPA, SB203580, SP600125, 5-Fluorouracil (5-FU), and Lucifer yellow were purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA). The working concentrations of the inhibitors were as follows: 25 μM SB203580 (Sigma-Aldrich), or 10 μM SP600125 (Sigma-Aldrich) 3 (link). Cells were pretreated with various inhibitors for 1 h, then EPA (100 μM) was added to cells for 24 h. Male C57BL/6 mice aged 6-8 weeks were purchased from the National Laboratory Animal Center of Taiwan. Adulthood is biologically defined as the age at which human or other organisms reach sexual maturity. Mice come of mating age sometime between 6-8 weeks of age. Adult mice 6-20 weeks old are the best to carryout studies 15 (link), 16 (link). The animals were maintained in specific pathogen-free animal care facility under isothermal conditions with regular photoperiods. The experimental protocol adhered to the rules of the Animal Protection Act of Taiwan and was approved by the Laboratory Animal Care and Use Committee of the National Sun Yat-sen University (the permit number: 10714).
Modified Eagle Medium (DMEM) with 1.5 g/L sodium bicarbonate, 25 mM glucose, and 10% fetal bovine serum (FBS), as previously described 14 . EPA, SB203580, SP600125, 5-Fluorouracil (5-FU), and Lucifer yellow were purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA). The working concentrations of the inhibitors were as follows: 25 μM SB203580 (Sigma-Aldrich), or 10 μM SP600125 (Sigma-Aldrich) 3 (link). Cells were pretreated with various inhibitors for 1 h, then EPA (100 μM) was added to cells for 24 h. Male C57BL/6 mice aged 6-8 weeks were purchased from the National Laboratory Animal Center of Taiwan. Adulthood is biologically defined as the age at which human or other organisms reach sexual maturity. Mice come of mating age sometime between 6-8 weeks of age. Adult mice 6-20 weeks old are the best to carryout studies 15 (link), 16 (link). The animals were maintained in specific pathogen-free animal care facility under isothermal conditions with regular photoperiods. The experimental protocol adhered to the rules of the Animal Protection Act of Taiwan and was approved by the Laboratory Animal Care and Use Committee of the National Sun Yat-sen University (the permit number: 10714).
4
Hepatic Cell Line Cultivation and Manipulation
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The human non-tumor hepatic cell L02 and liver cancer cell lines HepG2 and Huh7 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HBV genome (pUC18-HBV1.2) transfected HepG2 (HepG2.215) was from the Peking University Hepatology Institute (Beijing, China). HBX deficiency HepG2.215 (HepG2.215ΔHBX) and HBX overexpression HepG2 (HepG2-HBX) were obtained as previously described and expression of HBX in the cells mentioned above was detected (23 (link),24 (link)). The cells were cultured in Dulbecco's Modified Eagle Medium (high glucose) (HyClone; GE Healthcare Life Sciences, Logan UT, USA) supplemented with penicillin-streptomycin and 10% fetal bovine serum (Serana Europe GmBh, Pessin, Germany) at 37°C in 5% CO2. For AKT, JNK or nuclear factor (NF)κB inhibitor treatment, 10 mM LY294002 (Sigma-Aldrich, Merck KGaA), 10 mM SP600125 (Sigma-Aldrich, Merck KGaA) and 5 mM BAY11-7082 (Sigma-Aldrich; Merck KGaA) in DMSO were administrated to the cells with a final concentration of 10 µM LY294002, 10 µM SP600125 and 5 µM BAY11-7082 respectively in 37°C and 5% CO2 atmosphere. After 24 h, the cells were washed twice with PBS and collected for subsequent detection. In the cell viability assay, LY294002 was administrated immediately after cell adherence and maintained to the end.
5
Syk-mediated Signaling Pathway Modulation
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OREC were pretreated with the Syk-specific inhibitor R406 (1 μM and 5 μM) (InvivoGen, Toulouse, France) for 30 min, and the two different treatment groups were then stimulated with mannan. Cells stimulated with mannan, but not R406, represented the positive control. Cells treated with R406, but not mannan, represented the negative control, while untreated cells represented the blank control.
Then, the downstream pathways were inhibited using specific inhibitors: SB202190 (20 μM, Sigma) for p38, PD98059 (20 μM, Sigma) for ERK1/2, SP600125 (20 μM, Sigma) for JNK, and PDTC (10 μM, Sigma) for NF-κB. The cells were treated with the inhibitor for 60 min and the four different treatment groups were then stimulated with mannan (SB202190 + mannan, PD98059 + mannan, SP600125 + mannan, PDTC + mannan). Cells treated with only inhibitors (SB202190/PD98059/SP600125/PDTC) represented negative controls. Cells treated with mannan, but not inhibitors, represented the positive control, and untreated cells represented the blank control.
Then, the downstream pathways were inhibited using specific inhibitors: SB202190 (20 μM, Sigma) for p38, PD98059 (20 μM, Sigma) for ERK1/2, SP600125 (20 μM, Sigma) for JNK, and PDTC (10 μM, Sigma) for NF-κB. The cells were treated with the inhibitor for 60 min and the four different treatment groups were then stimulated with mannan (SB202190 + mannan, PD98059 + mannan, SP600125 + mannan, PDTC + mannan). Cells treated with only inhibitors (SB202190/PD98059/SP600125/PDTC) represented negative controls. Cells treated with mannan, but not inhibitors, represented the positive control, and untreated cells represented the blank control.
6
Modulating Inflammation in Colorectal Cancer
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For SP600125 treatment, WT and Rip3-/- mice were administered the JNK inhibitor SP600125 (40 mg/kg) (Sigma-Aldrich) or vehicle (DMSO) by oral gavage once daily during CAC induction. For anti-CXCL1 treatment, WT and Rip3-/- mice receiving AOM/DSS were treated either with a mouse anti-CXCL1 neutralizing antibody (120 µg/mouse once a week, i.p.) or mouse IgG1 isotype control (both from R&D Systems) until sacrifice (day 100). For anti-CD90 treatment, T cells were depleted with neutralizing anti-CD90 monoclonal antibody as previously described 23 (link). Rip3-/- mice receiving AOM/DSS were treated either with a mouse anti-CD90 neutralizing antibody (100 µg/mouse once a week, i.p.) or isotype control (both from BioXcell) until sacrifice (day 100). For recombinant CXCL1 treatment, Rip3-/- mice receiving AOM/DSS were treated either with mouse recombinant CXCL1 (300 ng/mouse twice a week, i.p.) or control PBS (both from R&D Systems) until sacrifice (day 100).
7
Investigating Endoplasmic Reticulum Stress in HepG2 Cells
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Human hepatoma (HepG2) cells (ATCC, Mannasas, VA) were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum and maintained at 37°C in 5% CO2. Cells were grown to 80% confluence in 6-well plates and treated with 12 μmol/L tunicamycin, 100 nmol/L thapsigargin (Sigma-Aldrich, St. Louis, MO), 5 mmol/L DL-homocysteine (Sigma-Aldrich), or vehicle (DMSO/saline) in serum-free DMEM for 6 hours. To determine whether the effects of tunicamycin are dependent on c-Jun-N-terminal kinase (JNK) or extracellular signaling-regulated kinase (ERK), HepG2 cells were treated with the JNK inhibitor SP600125 (Sigma-Aldrich) at a concentration of 25 μmol/L or the MAPK/ERK kinase inhibitor PD184352 (Santa Cruz Biotechnology, Dallas, TX) at 1 μmol/L or vehicle (DMSO/saline) as previously described.26 (link) One hour after exposure to SP600125 or PD184352, cells were treated with tunicamycin (12 μmol/L; Sigma-Aldrich) in serum-free DMEM and incubated for an additional 6 hours. Successful inhibition of JNK and ERK activation was confirmed by Western blot analysis as previously described.26 (link)
8
Cellular Stress Response Pathways
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Cells were starved for 24 hours and stimulated with 10% FCS for 1 hour or 30 J/m2 UV radiation for 24 hours before cells were harvested in 1× Laemmli Sample Buffer. For taxol experiments, cells were stimulated with 1% DMSO or 20 nmol·L−1 of taxol for 24 hours before cells were harvested in 1× Laemmli Sample Buffer. For SP600125 (Sigma‐Aldrich) experiments, cells were pretreated with 30 μmol·L−1 of SP600125 for 1 hour prior to stimulation with 10% FCS or 30 J/m2 UV radiation.
9
JNK Inhibitor Effects on PBMC Responses
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One hour before the stimuli addition, some of PBMC cultures were pre-treated with an inhibitor of JNK kinase. For inhibition of c-jun N-terminal kinase, we used the selective anthrapyrazolone inhibitor SP600125 (Sigma-Aldrich-Merck, Darmstadt, Germany). It competitively inhibits JNK 1, 2 and 3 with > 20-fold selectivity vs the wide range of kinases according to Bennett et al., [1 (link)]. SP600125 is dissolved in 100% dimethylsulfoxide (DMSO) (Sigma-Aldrich-Merck, Darmstadt, Germany), and the final concentration of JNK inhibitor in cell cultures is 20 µM. Non-stimulated cell cultures were used as controls. Therefore, our experiment included the following PBMC cultures: non-stimulated, stimulated with LPS, stimulated with LPS and pre-treated with SP600125, stimulated with C3bgp, stimulated with C3bgp and pre-treated with SP600125.
10
Investigating JNK Signaling Pathway
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LPS, PLA, and SP600125 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide, which was also purchased from Sigma-Aldrich, was used as a solvent for SP600125. Fetal bovine serum was purchased from Life Technologies Corporation (Carlsbad, CA, USA). Anti-JNK was purchased from Zhong Shan-Golden Bridge (Beijing, China). Anti-phospho-JNK and anti-β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Chemiluminescence reagents were purchased from Thermo Scientific (Rockford, IL, USA). ELISA kits for the detection of IL-6 and IL-8 were purchased from Senxiong (Shanghai, China).
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