INFH-hBMSCs were seeded at 1x103 in a 6-well plate, and randomly divided into four groups: LRRC17 overexpression treatment with or without 100 ng/ml DKK1 (MedChemExpress) treatment, and LRRC17-knockout cells with or without 10 µM SP600125 (Beyotime Institute of Biotechnology). Then, cells were transfected for the LRRC17 overexpression or knockout cells at 24 h after plating, followed by DKK1 or SP600125 treatment after 48 h. Cells were harvested after 24 h for downstream analysis.
Sp600125
Manufactured by Beyotime
Sourced in China, United States
SP600125 is a lab reagent that functions as a specific inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway. It acts by blocking the enzymatic activity of JNK, a family of mitogen-activated protein kinases involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by Beyotime (31 mentions)
U0126, by Beyotime (19 mentions)
Bay 11 7082, by Beyotime (11 mentions)
Pd98059, by Beyotime (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Beyotime (3 mentions)
N acetyl cysteine (nac), by Beyotime (3 mentions)
Ly294002, by Beyotime (3 mentions)
Cell counting kit 8 cck 8, by Beyotime (3 mentions)
Z vad fmk, by Beyotime (2 mentions)
β actin, by Beyotime (2 mentions)
Anti nf κb p65, by BioLegend (2 mentions)
Hrp conjugated anti mouse and anti goat igg, by Santa Cruz Biotechnology (2 mentions)
Phospho erk1 2, by BD (2 mentions)
P mtor, by Santa Cruz Biotechnology (2 mentions)
Antibody against lc3b, by Abcam (2 mentions)
P jnk, by Beyotime (2 mentions)
58 protocols using sp600125
1
LRRC17 Regulation of hBMSCs
2
Scutellarin-Breviscapine Cellular Effects
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Scutellarin (CAS#27740-01-8) was provided by Weiqi Biological Technology Co. Ltd. Breviscapine was provided by Yunnan Plant Pharmaceutical Co. Ltd. (National drug approval Z53020121). Z-VAD (OMe)-FMK (Z-VAD, HY-16658, MCE), SP600125 (S1876, Beyotime), CCK-8 kit (Japan Tongren Chemical Research Institute), Annexin V-FITC Apoptosis Detection Kit I (556547, BD Bioscience).
3
Reagents and Antibodies for Cell Assays
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Reagents. Neutral red staining solution was obtained from Sangon Biotech (Shanghai, China). U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) were purchased from Beyotime (Shanghai, China). LY294002 (PI3K inhibitor) was purchased from Selleck Chemicals (Houston, TX, USA). Anti-FIP-glu antiserum was raised in rabbits (99) . Anti-6×His Tag mouse monoclonal antibody, HRP-conjugated Goat Anti-Mouse IgG and HRP-conjugated Goat Anti-Rabbit IgG were from Sangon Biotech (Shanghai, China).
4
LPS-Induced Inflammatory Response in RAW 264.7 Cells
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RAW 264.7 cells were cultured in 6-well culture dishes until they reached approximately 80% confluence and were then stimulated with 1 μg/mL Escherichia coli LPS (InvivoGen, San Diego, CA, USA) for a certain number of hours. Cells not stimulated by LPS were used as a control.
In the signaling pathway inhibition experiments, the cells were first treated with the signaling pathway inhibitor BAY 11-7082 (Beyotime, Shanghai, China; 10 μM), U0126 (Beyotime, Shanghai, China; 10 μM), SB203580 (Beyotime, Shanghai, China; 20 μM) or SP600125 (Beyotime, Shanghai, China; 20 μM) for 1 h, and were then stimulated with 1 μg/mL LPS for 6 h. Cells not stimulated with LPS or treated with signaling pathway inhibitors were used as a blank control.
In the signaling pathway inhibition experiments, the cells were first treated with the signaling pathway inhibitor BAY 11-7082 (Beyotime, Shanghai, China; 10 μM), U0126 (Beyotime, Shanghai, China; 10 μM), SB203580 (Beyotime, Shanghai, China; 20 μM) or SP600125 (Beyotime, Shanghai, China; 20 μM) for 1 h, and were then stimulated with 1 μg/mL LPS for 6 h. Cells not stimulated with LPS or treated with signaling pathway inhibitors were used as a blank control.
5
Isolation and culture of VICs
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Valvular interstitial cells were isolated from patients undergoing Bentall surgery for acute aortic dissection, using the collagenase I digestion method previously described. 15 Briefly, isolated leaflets were digested in essential medium containing 1 mg/ml of collagenase type I at 37 C for 30 minutes. After removal of endothelial cells by vortexing, the leaflets were further digested with a fresh solution of 1 mg/ml of collagenase medium for 4 to 6 hours at 37 C. After vortexing and repeated aspirating to break up the tissue mass, the suspension was spun at 1000 rpm for 10 minutes to precipitate cells. Cells were resuspended and cultured in essential medium, supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum, in an incubator with 5% CO 2 , at 37 C. Cells of passages 2 to 5 were used for all experiments. When they had grown to 70% to 90% confluence, the VICs were stimulated with HMGB1 in the presence of the condition medium: 10 mmol of b-glycerophosphate; 100 nmol of dexamethasone; and 50 mg/ml ascorbic acid (all 3 from Sigma-Aldrich, St Louis, Mo). If needed, pharmacologic reagents, including 10 mmol/L SP600125 and 10 mmol/L BAY 11-7082 (both from Beyotime, Nantong, People's Republic of China), were added 1 hour before the addition of HMGB1. Cells from 3 patients were used for each intervention.
6
Bovine IL-1α Signaling Pathway Analysis
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Recombinant bovine IL-1α was purchased from Kingfisher Biotech. U0126, SP600125, SB203580 and BAY11-7082 were obtained from Beyotime Biotechnology. Erk antibody, p-Erk antibody, c-Jun antibody, p-c-Jun antibody, IκBα antibody and p-IκBα antibody were purchased from Cell Signaling Technology. p38 antibody, p-p38 antibody and β-actin antibody were purchased from LifeSpan BioSciences. Forskolin were purchased from Abcam.
7
Fluorescent Immunophenotyping of Dendritic Cells
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Fluorescent-labeled anti-mouse mAbs APC-CD11c (N418), PE-CD40 (1C10), PE-CD80 (16-10A1), FITC-CD86 (GL1), FITC-MHCII (M5/114.15.2), PE-CCR7 (4B12), FITC-CD4 (GK1.5), APC-IL-4 (11B11), PE-IFN-γ (XMG1.2), or respective isotype controls were purchased from eBioscience (San Diego, CA, USA). Mouse CD36 blocking antibody [JC63.1] and control IgA antibody were from Abcam (Cambridge, USA). Rabbit anti-mouse pERK, ERK, pJNK, JNK, pP38, P38, IκBα, p65, LaminB1, GAPDH, goat anti-rabbit IgG-DY488 and goat anti-rabbit IgG-HRP were from Bioworld (St. Louis Park, MN, USA). RPMI 1640 medium, DAPI, FITC-OVA, and CFSE were from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Hyclone (Thermo, Melbourne, Australia). Recombinant GM-CSF, IL-4, IL-2, and CCL19 were from Peprotech (Rocky Hill, NJ). LPS (from Escherichia coli 026: B6), 2′,7′-dichlorodi hydro fluorescein diacetate (DCFDA) and β-carotene were from Sigma-Aldrich (St Louis, MO, USA). PD 98, 059, SP600125, BAY 11-7082, Bradford assay kit, Nitric Oxide assay kit, Superoxide dismutase assay kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime (Jiangsu, China). Mouse IL-6, IL-10, IL-12p70, TNFα ELISA Kit and Cell Counting Kit-8 (CCK8) were from Boster (Wuhan, China).
8
Evaluating RPMTG's Effects on CRC Cell Lines
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The human CRC cell lines HCT116 and SW620 were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. HCT116 and SW620 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. The HCT116 (1×104 cells/well) and SW620 (2×104 cells/well) cells were pretreated with JNK inhibitor (SP600125, 10 µM; Beyotime Institute of Biotechnology) and p38 inhibitor (SB203580, 10 µM; Beyotime Institute of Biotechnology) for 2 h, and then treated with 250 µg/ml RPMTG for 24 h in a 37°C incubator containing 5% CO2. In subsequent experiments, the effects of RPMTG combined with JNK inhibitors or p38 inhibitors on proliferation and apoptosis were evaluated.
9
Asperolide A-Induced Apoptosis Regulation
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Asperolide A (AA) was dissolved with DMSO and stored at −20 °C, then diluted in cell culture medium. The final DMSO concentration is not more than 0.1%.
DMEM, fetal bovine serum and penicillin/streptomycin were purchased from Biowest (Maine et Loire, France). Annexin V-FITC/PI apoptosis detection kit, JC-1 detection kit, Cell cycle detection kit, and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were provided by Nanjing KeyGen Biotech Co. Ltd. (Nanjing, China). β-actin was from Tianjin Sungene Biotech (Tianjin, China). All the other antibodies were purchased from Cell Signaling Technology (Beverly, USA). DMSO, z-VAD-fmk, PD98059, SP600125 and SB203580 were provided by Beyotime Institute of Biotechnology (Shanghai, China).
DMEM, fetal bovine serum and penicillin/streptomycin were purchased from Biowest (Maine et Loire, France). Annexin V-FITC/PI apoptosis detection kit, JC-1 detection kit, Cell cycle detection kit, and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were provided by Nanjing KeyGen Biotech Co. Ltd. (Nanjing, China). β-actin was from Tianjin Sungene Biotech (Tianjin, China). All the other antibodies were purchased from Cell Signaling Technology (Beverly, USA). DMSO, z-VAD-fmk, PD98059, SP600125 and SB203580 were provided by Beyotime Institute of Biotechnology (Shanghai, China).
10
ApoE Isoform Effects on Axonal Regeneration
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Three human recombinant ApoE proteins, isoforms 2, 3 and 4 (PeproTech, Rocky Hill, NJ, USA), were separately added into Neurobasal/B27 to culture the explants of ApoE-/- mice after planting. The ApoE final concentration was 10 µg/ml in medium, which is similar to the concentration of ApoE in human cerebrospinal fluid (CSF)22 (link)–24 (link, link) . In addition, after axonal transection, the explants of the ApoE-/- mice were continuously cultured in Neurobasal/B27 with human recombinant ApoE2/3/4 proteins for 24 h.
JNK/ERK/p38 inhibitors, which included SP600125, U0126, and SB203580 (Beyotime, Shanghai, China), were also used as interventional means. The final concentration of the three inhibitors was 10 μM, and they were dissolved in dimethyl sulfoxide (DMSO, Beyotime)25 (link),26 (link). In the control group, only DMSO was added without the inhibitors. After explant planting, the explants were separately treated by medium with the JNK, ERK and p38 inhibitors for 24 h. Subsequently, the media were replaced with fresh media without the inhibitors27 (link).
JNK/ERK/p38 inhibitors, which included SP600125, U0126, and SB203580 (Beyotime, Shanghai, China), were also used as interventional means. The final concentration of the three inhibitors was 10 μM, and they were dissolved in dimethyl sulfoxide (DMSO, Beyotime)25 (link),26 (link). In the control group, only DMSO was added without the inhibitors. After explant planting, the explants were separately treated by medium with the JNK, ERK and p38 inhibitors for 24 h. Subsequently, the media were replaced with fresh media without the inhibitors27 (link).
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