Establishment and culturing of PDOs were performed according to Sato et al. 2011 [18 (link)]. Briefly, PDOs were cultured in Matrigel (Corning) using advanced DMEM/F12 medium supplemented with 10 mM Hepes, 1X Glutamax, 1X penicillin/streptomycin (10,000 U) (all from Gibco), 10 mM nicotinamide (Sigma), 10 µM SB202190 (MedChem Express, Monmouth Junction, USA), 1X B27 (Gibco), 1X N2 supplement (Gibco), 500 nM A83-01 (Tocris), 10% Noggin CM (made in house), 20% R-spondin conditioned media (CM) (made in house), 1.25 mM N-acetylcysteine (Sigma) and 50 ng/ml human EGF (Gibco). 100 µg/ml Primocin (InvivoGen) was added to the medium only during extracting and passaging PDOs for the first time. 10 µM Y-27632 (Adooq Biosciences, Irvine, USA) were added to the medium after extraction and seeding of PDOs. PDOs were respectively stratified for HGP based on H&E slides from the corresponding CRCLM.
Sb202190
Manufactured by MedChemExpress
Sourced in United States
SB202190 is a potent and selective inhibitor of p38 MAPK, a key enzyme involved in various cellular processes. It functions by blocking the phosphorylation and activation of p38 MAPK. SB202190 is commonly used in research applications to investigate the role of p38 MAPK in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Sp600125, by MedChemExpress (8 mentions)
U0126, by MedChemExpress (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Primocin, by InvivoGen (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modi ed eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Phenylmethanesulfonyl uoride pmsf, by Thermo Fisher Scientific (2 mentions)
Anti p38, by Abcam (2 mentions)
Anti p p38, by Abcam (2 mentions)
Anti erk1 2, by Abcam (2 mentions)
Anti jnk, by Abcam (2 mentions)
Anti p jnk, by Abcam (2 mentions)
Sybr green master mix, by Vazyme (2 mentions)
Hiscript reverse transcriptase, by Vazyme (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
18 protocols using sb202190
1
Establishment and Culture of Patient-Derived Organoids
2
Esophageal Cancer Cell Line Cultivation
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The ESCC cell lines KYSE2, KYSE180, KYSE450, KYSE510 and the human embryonic kidney cell line 293T (HEK293T) were obtained from the American Type Culture Collection (ATCC). ESCC cell lines were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). HEK293T was maintained in Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. Cells were collected and seeded in 6-, 24- or 96-well plates for 24 h and then transfected with plasmids or small interference RNAs (siRNAs) using Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA), according to the manufacturer's protocol. All experiments were conducted in cells with ∼80% convergence. The reagents used in this study were trichostatin A (TSA), cycloheximide (CHX), the inhibitors of Akt, ERK1/2 and p38, and aspirin (ASA). TSA and CHX were purchased from MedChem Express (USA). GSK690693 (an inhibitor of Akt), PD98059 (an inhibitor of the upstream kinase of ERK1/2), and SB202190 (an inhibitor of p38) were all purchased from MedChem Express (USA). ASA were purchased from Sigma-Aldrich (USA).
3
Reagent Acquisition and Purification
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LPS, ISO and NAC were obtained from Sigma (St. Louis, MO, USA); Tribromoethanol
was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI, USA) and SB202190
was purchased from MedChem Express (Monmouth Junction, NJ, USA). All other
chemicals were reagent grade and purchased from commercial sources and used
without further purification.
was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI, USA) and SB202190
was purchased from MedChem Express (Monmouth Junction, NJ, USA). All other
chemicals were reagent grade and purchased from commercial sources and used
without further purification.
4
Anti-inflammatory Effects of Crocetin
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Crocetin is donated by Tairui Biotechnology Co., Ltd (Nanyang, Henan, China). Crocetin was purified by HPLC with 98% purity, and it can be dissolved in DMSO (0.1% final concentration in culture medium). LPS (Escherichia coli Serotype 055:B5) was purchased from Sigma (St. Louis, MO, USA). AZD6244, SB202190, SP600125, and U0126 were from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against iNOS, phospho-ERK1/2, phospho-p38 kinase, phospho-JNK, ERK1/2, p38 kinase, JNK, and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH and p65 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against HO-1 and Nrf2 were from Abcam (Cambridge, MA, USA); 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was from Beyotime Institute of Biotechnology (Beyotime, Guangzhou, China). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz Beit Haemek, Israel). Murine macrophage-like RAW264.7 cells were purchased from ATCC (Rockville, MD, USA) and cultured at 37°C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. Because FBS contains numerous compounds, such as LPS and growth factors, which influence the biological characteristics of macrophages [38 (link)–41 (link)], we performed all of the experiments under serum-free conditions.
5
Esophageal Cancer Cell Line Cultivation and SFE Compound Preparation
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The human esophageal cancer EC109, KYSE510, KYSE150, and TE-1 cell lines were obtained from the National Infrastructure of Cell Line Resource, cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Gibco), 100 units/mL penicillin (Invitrogen, Carlsbad, CA, USA) and 100 mg/mL streptomycin (Invitrogen). The cell lines were characterized by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat markers and they were not contaminated by mycoplasma detected by Myco-Lumi Luminescent Mycoplasma Detection Kit (Beyotime, Shanghai, China). SFE was separated and purified from radish seeds (Beijing Tongrentang Co., LTD, Beijing, China) as reported previously2 (link),3 , dissolved in DMSO (Beijing Chemical Factory, Beijing, China). SB202190 was obtained from MedChem Express (Monmouth Junction, NJ, USA).
6
Establishing Long-term HGSC Organoid Cultures
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Long-term HGSC organoid cultures were established and characterized earlier as described (29 (link)). The samples were cultured in 7.5 mg/ml BME-2 matrix (Cultrex, BioTechne) in sample-specific media (29 (link))—for EOC883 and EOC172, Medium 1 [Advanced DMEM/F12 (#12634010, Gibco), supplemented with 100 μg/ml Primocin (#ant-pm-1, InvivoGen), 10 mM HEPES (#15630080, Gibco), 1 mM N-acetylcysteine (#A7250, Sigma), 1× GlutaMAX (#35050061), 1× B-27 Supplement, 0.5 μM SB202190 (#HY-10295, MedChemExpress), 0.5 μM A83-01 (#SML0788, Sigma), 10 ng/ml recombinant human FGF-10 (#100-26, PeproTech), 10 ng/ml recombinant human FGF-4 (#100-31, PeproTech), 100 nM β-estradiol (#E2758, Sigma) and 5 mM nicotinamide (#N0636, Sigma)]; for EOC989, EOC540 and EOC382, Medium 2 (Medium 1 supplemented with 5 ng/ml EGF, 5 μM heregulin-1β, 0.5 μg/ml hydrocortisone and 5 μM forskolin).
7
Osteogenic Differentiation of BMSCs
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Osteogenic differentiation was induced using standard osteoblast induction medium (10−8 M dexamethasone, 0.2 mM l-ascorbic acid, 10 mM, β-glycerophosphate and 10 mM 1.25-vitamin D3), as previously described (11 (link)). The JNK inhibitor, SP600125 (final concentration 20 µM; MedChemexpress, Princeton, NJ, USA), and the p38 inhibitor, SB202190 (final concentration 20 µM; MedChemexpress), were added to the BMSCs for 7 days. The BMSCs were fixed in 10% formalin for 30 min at room temperature and stained with Oil Red O (Beyotime, Jiangsu, China) to assess adipocyte differentiation. Subsequently, 85% propylene glycol was added to the cells for 5 min and the cells were observed under a microscope (LSM510 Meta Confocal Microscope; Carl Zeiss, Oberkochen, Germany).
8
Apoptosis and Autophagy Signaling Pathway
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The following reagents were used: Abs against LC3, ATG5, BAX, Bcl-2, caspase-8, and β-actin were purchased from Proteintech (CA, USA). Abs against JNK, phosphor-JNK, p38, phosphor-p38, ERK1/2, phosphor-ERK1/2, P62/SQSTM1 and cleaved caspase-3 were purchased from Cell Signaling Technology (Boston, USA). Abs against caspase-9 and poly (ADP-ribose) polymerases (PARP) were purchased from Abcam (Cambridge, United Kingdom). Goat anti-mouse IgG (H+L) cross-adsorbed secondary Ab Alexa Fluor 555 was purchased from Invitrogen (Massachusetts, USA). Immunofluorescence reagents were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). An Annexin V/PI kit was purchased from KeyGen Biotech (Jiangsu, China). Reactive oxygen species (ROS) and JC-1 kits were purchased from Thermo Fisher Scientific (Massachusetts, USA). CytoID autophagic detection kit was purchased from Enzo Life Sciences. Rapamycin (RAPA), 3-Methyladenine (3-MA), N-acetylcysteine (NAC) and SB-202190 were purchased from MedChemExpress (New Jersey, USA).
9
Impacts of MAPK Pathways on Pancreatic Cancer Migration
10
Investigating Stress-Induced Migration in Pancreatic Cancer
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To examine the role of p38 MAPK and JNK in the migratory ability of pancreatic cancer cells under 4.0 mmHg stress, MIA PaCa-2 and PANC-1 cells were grown in 2% FBS-containing DMEM for 24 hours and were then pretreated with 15 μmol/L of p38 MAPK inhibitor (SB202190, MedChemExpress) or JNK inhibitor (SP600125, MedChemExpress) for 1 hour. Control cells were pretreated with equal volume of solvent (DMSO). The concentration of each inhibitor was selected on the basis of previously published studies employing pancreatic cancer cells (22–24 (link)). Mechanical stress (4.0 mmHg) was then applied on cells growing in 2% FBS-containing medium in the presence of the inhibitors for 16 hours.
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