Primary cardiomyocytes were separated and maintained in DMEM containing 4.5 g/L of glucose and supplemented with 10% (v/v) FBS, and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 incubator. The cardiomyocytes were pretreated with 2.5, 5, and 10 µM emodin (E106693; Aladdin, Shanghai, China), 5 µM Bay-117082 (NF-κB pathway inhibitor) (S2913; Selleck, Shanghai, China), 10 µM NLRP3 inflammasome inhibitor (S3680; Selleck), or N-acetylcysteine (NAC, 1 mM, S0077; Beyotime, Shanghai, China) for 1 hour before the cardiomyocytes were stimulated with hypoxia/reoxygenation (H/R).
S2913
Manufactured by Selleck Chemicals
Sourced in China
S2913 is a laboratory equipment product from Selleck Chemicals. It is a device used for various scientific applications in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
S3680, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
S0077, by Beyotime (1 mentions)
Af 401 na, by R&D Systems (1 mentions)
Tlrl 3pelps, by InvivoGen (1 mentions)
Bp329 1, by Thermo Fisher Scientific (1 mentions)
Mp5 20f3, by R&D Systems (1 mentions)
Bay11 7082, by Selleck Chemicals (1 mentions)
Super tdu s8554, by Selleck Chemicals (1 mentions)
Chir 99021 ct99021, by Selleck Chemicals (1 mentions)
S2215, by Selleck Chemicals (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Thermo Fisher Scientific (1 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions)
4 protocols using s2913
1
Cardiomyocyte hypoxia/reoxygenation model
2
Recombinant Proteins and Endotoxin Assay
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Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems). This FGF23 peptide contains an arginine to glutamine amino acid substitution at position 179 which yields it resistant to furin protease-mediated degradation, thus prolonging its half-life. Lipopolysaccharide (LPS) from E. coli serotype 0111:B4 (tlrl-3pelps, Invivogen) was used as endotoxin. Sodium phosphate dibasic anhydrous (Na2HPO4) (BP332-500, Fisher Scientific) and sodium phosphate monobasic anhydrous (NaH2PO4) (BP329-1, Fisher Scientific) were used to prepare a 1 M stock sodium phosphate buffer solution containing 500 mM Na2HPO4 and 500 mM NaH2PO4 at an adjusted pH of 7.4. Sodium sulfate (Na2SO4) (239313, Sigma-Aldrich) was used to prepare a 1 M stock sodium sulfate buffer solution at an adjusted pH of 7.4. PFA (P6801, Sigma-Aldrich) and BAY 11-7082 (S2913, Selleckchem) were used as agents to elucidate underlying signal transduction mechanisms. Anti-IL6 (MP5-20F3, R&D Systems) and anti-IL1β (AF-401-NA, R&D Systems) were used as antibodies in a cell-based assay to neutralize the biological activity of targeted cytokines.
3
Agmatine Modulates Microglial Activation
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The mouse microglial cell line, BV2, was purchased from the German Collection of Microorganisms and cell cultures (DSMZ). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, United States) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37°C in 5% CO2. Preliminary experiments were performed to determine the dose of agmatine. BV2 microglial cells were pretreated with 1, 10, 50, 100, 200 or 500 μM agmatine. The dose chosen for subsequent experiments was 100 μM agmatine.5 µM Bay-117082 (an NF-κB pathway inhibitor) (S2913; Selleck, Shanghai, China), or a Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome inhibitor (10 µM, S3680; Selleck) was used to BV2 microglial cells for 1 h before LPS stimulation.
BV2 microglial cells were divided into four groups: the control group was administered sterile water; the AGM group was administered agmatine alone (100 μM); the LPS group was administered LPS (1 μg/ml) (Nam et al., 2018 (link)); and the LPS + AGM group was administered agmatine (100 μM) followed by LPS (1 μg/ml).
BV2 microglial cells were divided into four groups: the control group was administered sterile water; the AGM group was administered agmatine alone (100 μM); the LPS group was administered LPS (1 μg/ml) (Nam et al., 2018 (link)); and the LPS + AGM group was administered agmatine (100 μM) followed by LPS (1 μg/ml).
4
Recombinant CCN1 Protein Production
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Recombinant CCN1 and CCN1-D125A mutant proteins were produced using a baculovirus expression system in Sf9 insect cells and purified by ion exchange or immuno-affinity chromatography73 (link),74 (link). Sf9 cells (obtained from American Type Culture Collection, CRL-1711™) were routinely tested for mycoplasma contamination using LookOut® Mycoplasma PCR detection kit (Sigma). Purified CCN1 proteins were regularly examined for purity and contamination by SDS-gel electrophoresis and Limulus amoebocyte lysate method (for endotoxin) and also tested for specific activities in comparison with commercially available CCN1 protein (CCN1-Fc chimera protein; R&D systems, 4055-CR; Supplementary Fig. S5B ). Integrin αvβ3/αvβ5 inhibitor (SB273005; S7540), NF-κB inhibitor (Bay11-7082; S2913), γ-secretase inhibitor (DAPT; S2215), YAP-TEAD association inhibitors (Super-TDU; S8554, Verterporfin; S1786), FAK inhibitors (GSK2256098; S8523), and GSK3β inhibitor (CHIR99021; CT99021) were purchased from Selleckchem. Src inhibitor (Dasatinib; SML2589) was from Sigma–Aldrich. Recombinant Jag1 protein (599-JG) and Dkk1 protein (5897-DK) were from R&D systems. A neutralizing anti-Jag1 antibody (HMJ1-29) was from Invitrogen (16-3391-85) and an anti-Dkk1 antibody was from R&D systems (AF1096). 4′,6-diamidino-2-phenylindole (DAPI) solution (#62248) was from Thermo Fisher scientific.
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