Six days after differentiation, the dopaminergic-like in vitro model was exposed to culture conditions that mimic some PD mechanisms. The cells were exposed to different concentrations of 6-OHDA neurotoxin in culture media supplemented with 1% FBS for 24 h. 6-OHDA 25 µM induced about a 40% of cell death; thus, this concentration was selected to perform subsequent experiments. To study the effects induced by a PPAR β/δ antagonist (GSK0660, Sigma Aldrich, Saint Louis, USA) in our PD in vitro model, cells were co-treated with GSK0660 0.2 µM and 6-OHDA 25 µM. The in vitro model was exposed to GSK0660 in culture media supplemented with 1% FBS, 30 min later, 6-OHDA 25µM (diluted in culture media with 1% FBS) was added. Our in vitro model was maintained in these culture conditions for 24 h. Both 6-OHDA and GSK0660 stock solutions were prepared in Dimethyl sulfoxide and maintained at -20° C.
Gsk0660
Manufactured by Merck Group
Sourced in United States
GSK0660 is a small molecule compound developed by Merck Group for use in laboratory research. It is a selective agonist of the peroxisome proliferator-activated receptor gamma (PPAR-γ) nuclear receptor. The core function of GSK0660 is to activate PPAR-γ, which plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Gw9662, by Merck Group (4 mentions)
Gw0742, by Merck Group (4 mentions)
Gw6471, by Merck Group (2 mentions)
Telmisartan, by Merck Group (2 mentions)
Sodium nitrate, by Thermo Fisher Scientific (2 mentions)
Sulfanilamide, by Merck Group (2 mentions)
Orthophosphoric acid, by Thermo Fisher Scientific (2 mentions)
Lps o55 b5, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Gsk3787, by Merck Group (2 mentions)
L 165042, by Merck Group (2 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
Pdk 4, by Thermo Fisher Scientific (2 mentions)
Angptl4, by Thermo Fisher Scientific (2 mentions)
Anti phospho ampkα, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
15 protocols using gsk0660
1
Modeling Parkinson's Disease In Vitro
2
Investigating PPAR-α Signaling Pathways
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BZA was acquired from Sigma (B7273, purity > 98%). Phenylephrine (PE, P1240000) was obtained from Sigma-Aldrich. Anti-PPAR-α (sc-9000) and anti-PCNA (sc-7907) were purchased from Santa Cruz Biotechnology. The following first antibodies were purchased from Cell Signaling Technology: anti-AKT (#4691), anti-phospho-AKT (#4060), anti-GSK3β (#9315), anti-phospho-GSK3β (#9323P), anti-ERK (#4695), anti-phospho-ERK (#4370P), anti-P38 (#9212P), anti-phospho-P38 (#4511P), anti-AMPKα (#2603P), and anti-phospho-AMPKα (#2535). Anti-GAPDH (#ab8245), anticalcineurin (CaN) (#ab90540), and anti-NFAT1 (#ab2722) were obtained from ABCAM. Anti-α-actinin was acquired from Millipore. The secondary antibodies were purchased from LI-COR Biosciences. The PPAR-α antagonist (GW6471, G5045), PPAR-β/δ antagonist (GSK0660, G5797), and PPAR-γ antagonist (GW9662, M6191) were all purchased from Sigma-Aldrich. All other chemicals were of analytical grade.
3
Rh2 and PPAR-delta Antagonist Incubation
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When the cells reached 60% confluence, the culture was replaced with serum-free cell medium containing Rh2 (5 × 10–5 to 1 × 10–4 M; Tauto Biotech, Shanghai, China) with/without GSK0660 (a PPAR-delta antagonist, 1 × 10–6 to 5 × 10–6 M; Sigma-Aldrich, St. Louis, MO, USA). After drug incubation for 24 hours the cells were harvested by treatment with 0.25% trypsin and 0.2 g/L EDTA.
4
Molecular Mechanisms of PPARβ/δ Regulation
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GW0742 and GSK0660 were purchased from Sigma-Aldrich (St. Lois, MO, USA). Rosiglitazone, GW9662 and Bisphenol A diglycidyl ether (BADGE) are from Cayman Chemical Company (Ann Arbor, MI, USA). 5-bromo-2′deoxyuridine is from Sigma-Aldrich (St. Lois, MO, USA). Anti-PPARβ/δ and anti-Myc were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PPARγ, anti-GFAP (Glial fibrillary acidic protein), anti-DCX (Doublecortin) and anti-SOX2 are from Cell Signaling Technology (Beverly, MA, USA). Anti-Nestin and anti-EGFR are from Millipore (Billerica, MA, USA), anti-Galactocerebroside C (GalC) was purchased from Sigma-Aldrich (St. Lois, MO, USA), anti-5-bromo-2′deoxyuridine is from Abcam (Cambridge, MA, USA) and anti-β III-Tubulin is from Promega (Madison, WI, USA). Restriction enzymes are all from New England Biolabs (Ipswich, MA, USA). GoTaq Flexi DNA Polymerase and RT-PCR reagents were purchased from Promega (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA). siRNA-PPARβ/δ was purchased from Santa Cruz Biotechonology, siRNA-control and siGlo-Green Transfection Indicator were obtained from Thermo Fisher Scientific, Dharmacon Inc (Lafayette, CO, USA).
5
DMEM Protocol for Cell Culture Studies
6
Telmisartan and Losartan Effects on Metabolic Outcomes
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Once MS occurred, models and controls were treated by oral gavage with telmisartan (8 mg/kg/day; Boehringer Ingelheim, Ingelheim am Rhein, Germany)10 (link) or losartan (8 mg/kg/day; Zydus Pharmaceuticals, Pennington, NJ, USA) for 4 weeks. Moreover, PPARδ antagonist GSK0660 (10 mg/kg; Sigma-Aldrich Co., St Louis, MO, USA) was intraperitoneally injected 30 minutes before telmisartan administration.
Food and water intake were measured daily. Body weight was monitored weekly. BP was determined at week 9 (before drug treatment) and week 13 (the end of 4-week periods of the drug treatment) using the tail-cuff method with a sphygmomanometer (Muromachi Kikai Co., Ltd., Tokyo, Japan).33 (link)
At week 14, insulin tolerance tests (ITTs) were performed in the rats fasting overnight. According to a previous report,34 (link) rats were intraperitoneally injected with 0.75 IU/kg of regular insulin. Blood was collected from the tail vein of rats under anesthesia before injection and after 15, 30, 60, 90, and 120 minutes.
At the end of the study, livers and soleus muscles were collected from the sacrificed rats. The weight of retroperitoneal and epididymal fat pads were also measured. All samples were immediately frozen in liquid nitrogen and kept at –80°C for further assays.
Food and water intake were measured daily. Body weight was monitored weekly. BP was determined at week 9 (before drug treatment) and week 13 (the end of 4-week periods of the drug treatment) using the tail-cuff method with a sphygmomanometer (Muromachi Kikai Co., Ltd., Tokyo, Japan).33 (link)
At week 14, insulin tolerance tests (ITTs) were performed in the rats fasting overnight. According to a previous report,34 (link) rats were intraperitoneally injected with 0.75 IU/kg of regular insulin. Blood was collected from the tail vein of rats under anesthesia before injection and after 15, 30, 60, 90, and 120 minutes.
At the end of the study, livers and soleus muscles were collected from the sacrificed rats. The weight of retroperitoneal and epididymal fat pads were also measured. All samples were immediately frozen in liquid nitrogen and kept at –80°C for further assays.
7
Polydatin Attenuates Endothelial Dysfunction
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Polydatin (C20H22O8; MW: 390.38; purity ≥ 95%) and GSK0660 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polydatin was dissolved in 1% dimethysulfoxide (DMSO) and directly diluted in medium to the required concentrations before the experiments. Acetylcholine, phenylephrine (PE), NG-nitro-L-arginine methyl ester (L-NAME), meloxicam, horseradish peroxidase-conjugated goat anti-rabbit IgG and the nitrite detection kit were purchased from Beyotime (Jiang Su, China). RIPA lysis buffer, the BCA protein concentration assay kit (Enhanced), and the total NOS (tNOS) and eNOS detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Trizol and the reverse transcription kit were purchased from Takara BIO Inc (Otsu, Shiga, Japan). The anti-PPARβ, anti-eNOS, anti- inducible NOS (iNOS), anti-GADPH antibodies were purchased from Abcam (Cambridge, UK). All other reagents were of analytical grade. The gel imager and Quantity one software, the i-mark microplate reader and the icycler quantitative PCR instrument were purchased from BIO-RAD (Hercules, CA, USA).
8
Ligand-Induced PPARβ/δ Signaling
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The PPARβ/δ ligands GW0742, GSK3787, L−165042 and GSK0660 as well as 1400W, LPS O55:B5, sulfanilamide and naphthylethylenediamine dihydrochloride were purchased from Sigma (Gillingham, Dorset, UK). Sodium nitrate, orthophosphoric acid and DMSO were purchased from Fisher Scientific (Loughborough, UK). Primers from Applied Biosystem (Foster City, CA, USA): β-actin (Rn00667869_m1), Pdk−4 (Rn00585577_m1), Angptl−4 (Rn015228817_m1), Nos2 (Rn00561646_m1).
9
Lipid Signaling Pathway Regulation
10
Investigating HaCaT Cell Responses
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HaCaT cells and human keratinocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (AA) in 37°C and 5% CO2 incubator. The cell culture medium was replaced with new DMEM every 48-72 h. HaCaT cells within 5~17 passages were plated at a density of 1×104 cells per well in 8-well chamber slide, or 1×106 cells per well in a six-well culture plate in DMEM containing 10% FBS and 1% AA. The cells were cultured for 24 to 48 h in 5% CO2 incubator at 37°C. Then, the medium was changed to DMEM containing 1% FBS. Thereafter, the cells were treated by DNCB (5 μmol; Sigma-Aldrich, Louis, MO, USA) or SDS (30 μmol; Sigma-Aldrich) along with AG extract (50 μg) and PPARδ antagonist, GSK0660 (50 μmol; Sigma-Aldrich), AMPK antagonist, Compound C (1 μmol; Sigma-Aldrich), or TRPV4 antagonist, GSK2193874 (50 nmole; Sigma-Aldrich) for 24 hours. HaCaT cells were donated from Dr. Sang-Wook Son. All reagents for cell culture were purchased from WELGENE Inc. (Daegu, Republic of Korea). The treating concentrations of all reagents were final concentrations.
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