Wild-type male C57BL/6 J mice aged 6-8 weeks (approx. 22 g) were used to establish the model of AKI induced by I/R. All mice were provided by the animal house of the Central Laboratory of the Ninth Hospital of Shanghai Jiao Tong University. The experimental procedures were approved by the Ethics Committee of the Ninth People's Hospital (SH9H-2020-A655-1). Mice were equally divided into four groups: sham + normal saline (NS), I/R + NS, sham + 3-DZNeP, and I/R + 3-DZNeP. NS or 3-DZNeP (2 mg/kg, Selleck, USA) was intraperitoneally injected at 24 h before surgery. Mice were fasted for 12 h before the surgery. After anesthesia with 2% sodium pentobarbital (40 mg/kg), the abdomen of mouse was exposed, and both renal arteries were clamped for 30 min to achieve kidney ischemia. During this period, the animals were closely monitored to prevent a decrease in body temperature. After the ischemic period, the arterial clamps were removed. If the kidneys changed from dark red to bright red, it indicated a successful modeling. Then, 500 μL of sterile saline was added to the abdominal cavity, and the incision was sutured. The sham mice underwent the same surgical procedure without clamping the renal arteries. At 24 h after reperfusion, the mice were euthanized for blood and kidney collection.
3 dznep
Manufactured by Selleck Chemicals
Sourced in United States
3-DZNeP is a chemical compound used as a laboratory research tool. It functions as a selective inhibitor of the histone methyltransferase EZH2, which is involved in the regulation of gene expression. This product is intended for research purposes only, and its specific applications should be evaluated by trained professionals.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Cell Signaling Technology (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Scramble sirna, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Cisplatin, by Merck Group (1 mentions)
E cadherin sirna, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Antibodies to caspase 3, by Cell Signaling Technology (1 mentions)
Antibody to ngal, by R&D Systems (1 mentions)
Ezh2 sirna, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence ecl detection kit, by Beyotime (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Mmp 9, by Merck Group (1 mentions)
Mmp 2, by Santa Cruz Biotechnology (1 mentions)
Antibodies to e cadherin, by Santa Cruz Biotechnology (1 mentions)
P raf 1, by Santa Cruz Biotechnology (1 mentions)
5 protocols using 3 dznep
1
Murine Model of Ischemic Acute Kidney Injury
2
Molecular Mechanism of 3-DZNeP and Cisplatin
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3-DZNeP was purchased from Selleckchem (Houston, TX, USA). Cisplatin was purchased from Sigma (St. Louis, MO, USA). Antibodies to caspase3, E-cadherin, EZH2, H3K27me3, p-JNK, p-ERK1/2, p-p38, histone H3, and tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody to NGAL was purchased from R&D systems (Minneapolis, MN, USA). Antibodies to GAPDH, β-actin, horseradish peroxidase-conjugated secondary antibodies, enhanced chemiluminescence (ECL) detection kit, cell counting kit-8 (CCK-8), and DAPI were from Beyotime institute of Biotechnology (Jiangsu, China). Scr and BUN reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). EZH2 siRNA, E-cadherin siRNA and scramble siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
Comprehensive Antibody Panel for Cellular Signaling Investigations
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Antibodies to EZH2, H3K27me3, RKIP, cleaved Caspase-3, and P-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to E-cadherin, MMP-2, P-Raf-1, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved PARP (24 kDA) and TIMP-2 antibodies were purchased from Abcam, Inc. (Cambridge, MA). Neutrophil gelatinase-associated lipocalin NGAL antibody was obtained from R&D Systems (Minneapolis, MN). ZO-1, MMP-9, TIMP-3 antibodies were purchased from EMD Millipore (Billerica, MA). 3-DZNeP was purchased from Selleck Chemicals (Houston, TX). ɑ-tubulin and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). SiRNA specific for EZH2 was obtained from Invitrogen (Carlsbad, CA). All other antibodies used in this study were purchased from Cell Signaling Technology (Danvers, MA).
4
Polarization of Alveolar Macrophages and EZH2 Modulation
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MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin in an atmosphere of 5% CO2, and 95% air at 37℃. According to the previous report [18 ], MH-S cells were polarized as M1 or M2 macrophages with the indicated stimulants: 50 ng/mL LPS (L3024, Sigma, Mo) 24 h for M1 polarization, and 10 ng/mL IL-4 (404-ML, R&D Systems) 24 h for M2 polarization. The 3-DZNeP (Selleckchem, Houston, TX, USA) (0, 1, 2, 5 and 10 μM) was added 24 h before LPS or IL-4 stimulation. The small interfering (si) RNA oligonucleotides targeted to EZH2 (GenePharma, Shanghai, PR China) were used to knock down EZH2 more specifically in vitro. The MH-S cells were transfected with EZH2 siRNA (100 pmol) with lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) or scrambled siRNA (100 pmol) according to the manufacturer’s instructions 24 h before LPS or IL-4 stimulation. After different stimulations, cells and supernatants were harvested for analyses. All of the in vitro experiments were repeated at least three times.
5
3-DZNeP Attenuates Sepsis-Induced AKI
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To study the effect of 3-deazaneplanocin A(3-DZNeP) (Selleckchem, Houston, USA) on sepsis-induced AKI, after injecting LPS or CLP, 3-DZNeP (2 mg/kg) was delivered intraperitoneally every day after that. Equal amounts of saline were injected into the control group. On the third day following the injection of LPS or CLP, the mice were sacrificed.
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