Celastrol (No. PS0048‐0020, purity ≥ 98%) was purchased from Push Bio‐Technology Co., Ltd. (Chengdu, China). Primary antibodies against anti‐rabbit α‐SMA (14395‐1‐AP), α(I) procollagen (14395‐1‐AP), fibronectin (15613‐1‐AP), PPARγ (16643‐1‐AP) and GAPDH (13937‐1‐AP) were purchased from proteintech. Primary antibodies against SIRT3 (#5490), p‐AMPK (#2537) and AMPK (#2532) were purchased from Cell Signalling Technology (Danvers, MA, USA). Primary antibody against PGC‐1α was purchased from Affinity (AF5395). Hydroxyproline examination kit (A030‐2‐1) was purchased from Nanjing Jiancheng Bioengineering Institute. ELISA kits including IL‐6 (H007), IL‐18 (H015), IL‐1β (H002), TNF‐α (H052), IFN‐γ (H025) and IL‐10 (H009) were purchased from Nanjing Jiancheng Bioengineering Institute. Dorsomorphin (Compound C, an AMPK inhibitor) (S7840) was purchased from Selleck. The primers used in real‐time PCR were from GenScript Co. Ltd. MegaTran 1.0 transfection reagent was from OriGene. SIRT3 enzyme activity detection kit (JK50288.2) was purchased from Shanghai Baoman Biotechnology Co., Ltd.
Dorsomorphin compound c
Manufactured by Selleck Chemicals
Sourced in United States
Dorsomorphin (Compound C) is a synthetic small molecule that functions as a selective inhibitor of AMP-activated protein kinase (AMPK). It is commonly used in research applications to study AMPK signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Selleck Chemicals (2 mentions)
Megatran 1.0 transfection reagent, by OriGene (1 mentions)
Il 10, by Nanjing Jiancheng (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Metformin, by Merck Group (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Anti p21, by Proteintech (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Ab110330, by Abcam (1 mentions)
Anti flag af0036, by Beyotime (1 mentions)
Anti phb2, by Proteintech (1 mentions)
Anti phb, by Proteintech (1 mentions)
Gsk621, by Selleck Chemicals (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti p53, by Proteintech (1 mentions)
Canagliflozin, by Selleck Chemicals (1 mentions)
Acadesine aicar, by Selleck Chemicals (1 mentions)
D glucose, by Procell (1 mentions)
14 protocols using dorsomorphin compound c
1
Celastrol Modulates SIRT3-AMPK Signaling in Liver Fibrosis
2
Culturing and Transfecting Cell Lines
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The Raji, K562, and 293FT cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The Raji cells were routinely cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin in 5% CO2 at 37°C. Raji-ffluc cells for bioluminescent imaging were generated by transfecting Raji cells with an expression cassette for firefly luciferase. The K562 cells were cultured in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin. 293FT cells were maintained in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) supplemented with 10% FBS. All cells were cultured at in 5% CO2 at 37°C. Metformin was purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and then dissolved in PBS. Dorsomorphin (Compound C), a potent AMPK inhibitor, was purchased from Selleck (Houston, TX, USA) and then dissolved in dimethyl sulfoxide.
3
Protein Purification and Detection Assay
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PS-341 (cat. no. 1846-1) was purchased from BioVison. Cycloheximide (CHX, C8030–100) was purchased from Solarbio. Doxorubicin hydrochloride (D1515-10MG) was purchased from Sigma-Aldrich. Dorsomorphin (Compound C) and GSK621 were obtained from Selleck. Antibodies used in this study were listed with the source in parentheses - anti-QCR2 (14742-1-AP, Proteintech), anti-GAPDH (10494-1-AP, Proteintech), anti-p53 (10442–1-AP, Proteintech), anti-p21 (10355-1-AP, Proteintech), anti-Flag (AF0036, Beyotime), anti-PHB (10787-1-AP, Proteintech), anti-Ubiquitin (BML-PW8390-0100, Enzo), anti-PHDA1 (ab110330, Abcam), anti- AMPK (5831T, CST), anti-p-AMPK (2535T, CST), anti-PCNA (10205-2-AP, Proteintech), anti-α-Tubulin (11224-1-AP, Proteintech), anti-PHB2 (12295-1-AP, Proteintech). Flag Agarose (PM020-8) used for immunoprecipitation was obtained from Medical & Biological Laboratories.
4
MC3T3-E1 Osteoblast Differentiation Protocol
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MC3T3-E1 Subclone 14 cells were purchased from Procell company (Wuhan, China) (RRID : CVCL_5437) and were cultured with α-mem medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and a normal glucose concentration (NG) of 5.5 mM. For high-glucose treatment, the culture medium mentioned contained a 33 mM glucose concentration supplied with D-glucose (Procell company, Wuhan, China), and the cells were treated for 7 days prior to performing the experiments. Cells were seeded on 6-well or 12-well plates in complete medium for 12 h. Canagliflozin (Selleck Chemicals, Houston, TX), a p-AMPK inhibitor dorsomorphin (compound C, Selleck Chemicals, Houston, TX), or an AMPK activator, acadesine (AICAR, Selleck Chemicals, Houston, TX) was added and removed after incubation for 12 h. Then, MC3T3-E1 cells were induced with osteogenic inductive medium (Procell company, Wuhan, China) on the indicated days to perform ALP staining, Alizarin red staining, and protein collection.
5
Netrin-1 Neuroprotection Signaling Pathway
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Recombinant rat Netrin-1 was purchased from Creative Biomart (Shirley, NY, USA). Anti-LC3B, anti-Beclin-1, anti-NeuN, anti-mTOR, and anti-p-mTOR antibody as well as goat anti-rabbit and goat anti-mouse-IgG HRP were purchased from Abcam (Cambridge, MA, USA). Anti-AMPK, anti-p-AMPK, anti-p-P70S6K, anti-P70S6K, and anti-caspase-3 antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor® 568 and Alexa Fluor® 488 were acquired from Life Technology (Carlsbad, CA, USA). Anti-β-tubulin antibody was provided by TransGen Biotech (Beijing, China). Dorsomorphin (Compound C), an AMPK inhibitor, was supplied by Selleck Chemicals LLC (Houston, TX, USA). An enhanced chemiluminescence (ECL) kit was obtained from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). In situ Cell Death Detection Kit was purchased from Roche (Mannheim, Germany). All of the other reagents were acquired from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified.
6
Isolation and Activation of Murine Regulatory T Cells
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Dexmedetomidine (Sigma-Aldrich, SML0956); Dorsomorphin (Compound C) (Selleck, S7306); Cell Counting Kit8 (MCE, HY-K0301); Human lymphocyte separation solution (GE Healthcare, FICOLL-PAQUE PLUS, 17,144,003); EasySep™ Mouse Naive CD4+ T Cell Isolation Kit (Stem cell, 19,765); IL-2 (Peprotech, 212–12); TGF-β1 (Peprotech, 100–21); Anti-Mouse CD3 antibody (eBioscience, 16–0032-86), Anti-Mouse CD28 antibody (eBioscience, 16–0281-85); Anti-Mouse CD25 antibody (eBioscience, 16–0251-38); CD4 Monoclonal antibody (GK1.5), FITC (eBioscience, 11–0041-82); Rat IgG2b kappa Isotype Control (eB149/10H5), FITC (eBioscience, 11–4031-82); CD25 Monoclonal Antibody (PC61.5), PE-Cyanine7 (eBioscience, 25–0251-82); Rat IgG1 kappa Isotype Control (eBRG1), PE-Cyanine7 (eBioscience, 25–4301-82); HELIOS Monoclonal Antibody (22F6), PE (eBioscience, 12–9883-42); Armenian Hamster IgG Isotype Control (eBio299Arm), PE (eBioscience, 12–4888-81); FOXP3 Monoclonal Antibody (FJK-16 s), APC (eBioscience, 17–5773-82); Rat IgG2a kappa Isotype Control (eBR2a), APC (eBioscience, 17–4321-81). Since the antibodies used in immunohistochemistry, immunofluorescence, and western blotting experiments are not identical, information on these antibodies will be described in the corresponding methods section.
7
Exploring PPAR and AMPK Modulators
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PPARβ/δ selective agonist GW501516 and PPARβ/δ antagonist GSK3787 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The AMPK agonist AICAR and inhibitor dorsomorphin (compound C) were bought from Selleck Chemical (Houston, TX, USA).
8
Evaluating Metabolic Modulators in Cell Experiments
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VAC was purchased from Shanghai Shifeng Technology (Shanghai, China). N-acetyl-L-cysteine (NAC) was obtained from Beyotime Institute of Biotechnology (Shanghai, China). 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) and Streptozocin (STZ) were acquired from Sigma (St. Louis, MO, USA). Dorsomorphin (Compound C) (S7840) was purchased from Selleck (Houston, TX, USA). Valproate sodium (VPA) was purchased from Med Chem Express (Monmouth Junction, NJ, USA). AMPK, p-AMPK, β-actin antibody were bought from Abcam (Cambridge, MA, USA). eNOS, p-eNOS (Ser1177) antibody were bought from Abclonal (Wuhan, China). The specific primers were synthesized by Shanghai Sangon Biotech Co. Ltd. (Shanghai, China). Human NO Production Kit was purchased from Shanghai Yuanmu Biological Technology Co. Ltd. (Shanghai, China).
9
Modulation of Adipocyte Response to TNFα
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AICAR (CAS No. 2627-69-2, PubChem CID: 17513) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Recombinant mouse TNFα (expressed in Escherichia coli), dimethylbiguanide hydrochloride (metformin), LY294002, and pyrrolidine dithiocarbamate (PDTC) were from Sigma Chemical Co. (St. Lois, MO, USA). Dorsomorphin (compound C) was purchased from Selleck Chemicals (Houston, TX, USA).
3T3-L1 cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK, No. 86052701), derived from the mouse embryonic fibroblast cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% bovine serum (Gibco BRL, Rockville, MD, USA) and were differentiated as described previously6 (link)–10 (link)). The cells were checked for the degree of differentiation microscopically. Well-differentiated (>90%) 3T3-L1 adipocytes were exposed to TNFα (1–100 ng/ml) for 24 h. AICAR and/or other reagents were added 1 h before the TNFα treatment. As controls, 3T3-L1 adipocytes were cultured without any treatments for the same length of time.
3T3-L1 cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK, No. 86052701), derived from the mouse embryonic fibroblast cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% bovine serum (Gibco BRL, Rockville, MD, USA) and were differentiated as described previously6 (link)–10 (link)). The cells were checked for the degree of differentiation microscopically. Well-differentiated (>90%) 3T3-L1 adipocytes were exposed to TNFα (1–100 ng/ml) for 24 h. AICAR and/or other reagents were added 1 h before the TNFα treatment. As controls, 3T3-L1 adipocytes were cultured without any treatments for the same length of time.
10
Novel Fluorescent Glucose Uptake Assay
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2-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-D-glucosamine (2-NBDG; catalog number: N13195) was purchased from Invitrogen™ (Waltham, MA, USA). Molecular biology-grade DMSO was purchased from Sigma-Aldrich (St. Louis, MO, USA). D. perchlorate (batch number: X-059-181114) was purchased from Chengdu Herb Purify Co., Ltd. (Chengdu, China). Dragon blood (commercial name: Draconis Sanguis; batch number: 712,041), containing 1.3% dracorhodin, was obtained from Ledstream Technology SDN BHD (Pulau Pinang, Malaysia). Oillio quick thickener was obtained from Shun Hwa Pharmaceutical (Taipei City, Taiwan). The inhibitors, LY294002 (catalog number: S1105) and dorsomorphin (Compound C, catalog number: S7840), were purchased from Selleck Chemicals, LLC. (Houston, TX, USA).
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