Cells were seeded in 12-well plates for cell viability or in P100 plates (Corning, New York, NY, USA) for RNA and protein purification at a density of 100.000 cells/well or 1.2 × 106 cells/plate, respectively. Then, cells were allowed to attach and reach 100% confluency for 24 h before removing the medium and replacing it with serum-free medium for 24 h. Treatments were then added and maintained for 24 h, with the exception of ELISAs that were maintained for 48 h, and some protein extracts that were collected at shorter times. Cells were treated with 8–20 μM 7KCh (Sigma-Aldrich, Madrid, Spain) alone or with 10 μM SA (PPQF, University of Alcalá, Madrid, Spain), 10 μM of the TLR4 inhibitor CLI-095 (Invivogen Inc., San Diego, CA, USA), 10–50 μM of the JNK inhibitor SP600125 (StressMarq Biosciences Inc., Victoria, BC, Canada) or 5–40 μM of the p38 inhibitor SB203580 (Sigma-Aldrich, Madrid, Spain). 7KCh was prepared in β-cyclodextrin (Sigma-Aldrich, Madrid, Spain) as previously described [15 (link),20 (link)], CLI-095 was dissolved in culture medium and SA, SP600125 and SB203580 were dissolved in DMSO (Sigma-Aldrich, Madrid, Spain).
Sb203580
Manufactured by Merck Group
Sourced in United States, Germany, Macao, United Kingdom, China, Japan, Spain, Switzerland, Canada, Sao Tome and Principe, Sweden, Italy
SB203580 is a lab equipment product manufactured by Merck Group. It is a pyridinyl imidazole compound that functions as a selective inhibitor of p38 mitogen-activated protein kinase (MAPK).
Automatically generated - may contain errors
Lab products found in correlation
Sp600125, by Merck Group (221 mentions)
Pd98059, by Merck Group (157 mentions)
Ly294002, by Merck Group (102 mentions)
U0126, by Merck Group (94 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (72 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (37 mentions)
Bay11 7082, by Merck Group (36 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
Sb202190, by Merck Group (21 mentions)
Wortmannin, by Merck Group (20 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (18 mentions)
β actin, by Cell Signaling Technology (17 mentions)
β actin, by Santa Cruz Biotechnology (17 mentions)
U0126, by Cell Signaling Technology (16 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (16 mentions)
Mg132, by Merck Group (15 mentions)
Gapdh, by Cell Signaling Technology (14 mentions)
Lipopolysaccharides (lps), by Merck Group (14 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Sb431542, by Merck Group (13 mentions)
696 protocols using sb203580
1
7KCh Cytotoxicity Pathway Inhibition
2
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).
3
Inhibition of p38α/β and NADPH Oxidase in SCC
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A431 human epidermoid squamous cell carcinoma (SCC) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained according to ATCC guidelines. Primary human keratinocytes isolated from neonatal foreskin were obtained from Columbia University Skin Disease Research Center (SDRC) tissue culture core facility and cultured in 154CF medium supplemented with human keratinocyte growth supplement (Life Technologies, Grand Island, NY). SB203580, a pyridinyl imidazole inhibitor widely used to inhibit the biological function of p38α/β [26] (link)–[28] (link), and diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase-mediated ROS formation, were purchased from Sigma-Aldrich (St. Louis, MO). siRNA targeting p38α coding regions and scrambled siRNA controls (con) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). SB203580 was dissolved in DMSO for cell culture or in 0.5% methylcellulose (Sigma-Aldrich) for oral administration to mice. DPI was dissolved in dimethyl sulfoxide (DMSO) and added to cell cultures at a final concentration of 2.5 µM. SCC tissue microarrays (TMAs) were obtained from Imgenex (San Diego, CA).
4
Organoid Viability: Vitamin, Inhibitor Effects
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To evaluate the effects of vitamin A, vitamin D, signal transducers and activators of transcription 5 (STAT5)-inhibitor STAT5-IN-1, or p38 mitogen-activated protein kinase (p38 MAPK)-inhibitor SB203 580 (5 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany) on organoid cell viability, an MTT reduction assay was performed. For this purpose, organoids were incubated with calcitriol (50 nM, 100 nM, 200 nM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with retinoic acid (10 µM, 100 µM, 200 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with STAT5-IN-1 (5 µM, 0.5 mM, 1 mM, solved in DMSO; Selleckchem, Planegg, Germany), or with SB203 580 (5 µM, solved in DMSO; Sigma-Aldrich, Darmstadt, Germany), or with a corresponding amount of DMSO as control for 30 h. Then, MTT solution (500 µg/mL; solved in PBSO; Merck, Darmstadt, Germany) was added to organoids to a final concentration of 500 µg/mL. After incubation for 1 h at 37 °C, 5% CO2, CCM was discarded and cells were incubated with 20 µL of SDS (for 1 h at 37 °C, 5% CO2) to dissolve Matrigel. Finally, 100 µL of DMSO was added (for 1 h at 37 °C, 5% CO2) to dissolve reduced MTT, and the optical density was measured at 562 nm on a microplate absorbance reader (BioTek Instruments, Winooski, VT, USA).
5
Cigarette Smoke Extract Cytotoxicity
6
Macrophage Polarization and p38 MAPK Inhibition
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BMDMs were isolated from 7- to 8-week-old mice and cultured as described previously to obtain naïve M0 macrophages (Dai et al., 2017 (link)). For M1 polarization, BMDMs were stimulated with 10 ng/ml LPS (Sigma Aldrich) for 24 h. For M2 polarization, BMDMs were stimulated with 10 ng/ml IL-4 (BioLegend) for 24 h. Pharmacologic inhibition of p38 MAPK (SB203580; Sigma Aldrich) was achieved by pretreating BMDMs with 10 µM SB203580 for 1 h before addition of LPS or IL-4.
7
Investigating Cellular Responses to Nanoparticles
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For ODN2088 (Invitrogen), in vivo ODN2088 (50 μg/mouse) was administrated via tail vein prior to instillation of mtDNA, and in vitro ODN2088 (25 μg) was added in the medium prior to mtDNA treatment. For CA074 Me (Millipore), in vitro MH-S cells were pre-treated with CA074 Me (100 μM, 40 μg) for 30 min before stimulation and then treated with CBNPs (100 µg/cm2) for 30 min with the presence of CA074 Me, and in vivo CA074 Me (40 μg/mouse) was administrated via tail vein 2 h prior to instillation of CBNPs. For SB 203580 (Sigma), in vivo SB 203580 (3.77 μg/mouse) was administrated via tail vein prior to instillation of CBNPs.
8
Nmnat1 Expression and MAPK Inhibition in RGC-5 Cells
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RGC-5 cells were transfected with Nmnat1 siRNA or Nmnat1 plasmid to change Nmnat1 expression levels. After that, RGC-5 cells were pretreated with MAPK inhibitors (U0126, 10 µM; SB203580, 10 µM; SP600125, 10 µM) for 1 h to inhibit MAPK signaling, and then exposed to high glucose (30 mM) for 48 h. U0126 (ERK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were purchased from Sigma Chemical.
9
Inhibiting p38 in Imiquimod-induced Inflammation
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Imiquimod-treated WT and MKP-1−/− mice were intraperitoneally administrated with p38 inhibitor SB203580 (Merck CalBiochem) at a dose of 0.75 mg/kg body weight for five consecutive days. For in vitro macrophage treatment, cells were incubated with vehicle or 10 μM SB203580 (Merck Calbiochem) for 0.5 h before stimulation.
10
Osteogenic Induction of Dental Pulp Stem Cells
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The osteogenic induction medium was composed of DMEM, 10−8 M dexamethasone, 50 mg/ml L-ascorbic acid, 0.005 M KH2PO4, 50 mg/ml gentamycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and 10% FBS. The medium was replaced every 3 days for 3 weeks. The conditions used in the different experimental groups were as follows: Control group, DPSCs cultured in DMEM with 10% FBS; Dx group, DPSCs cultured in the osteogenic induction medium dexamethasone (Dx); Dx-PD98059 group, DPSCs cultured in Dx medium with 10 µM PD98059, an inhibitor of Erk1/2; and Dx-SB203580 group, DPSCs cultured in Dx medium with 10 µM SB203580, an inhibitor of p38. The inhibitors, PD98059 (Merck KGaA) and SB203580 (Merck KGaA), were added to the DMEM medium 2 h prior to the shift to osteogenic induction medium (also including PD98059 and SB203580) for continuous culture.
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