AML-12 mouse hepatocytes were cultured as instructed. Cells were cultured at 37 °C with 5% CO2 in a humidified atmosphere. After 24 h serum starvation, cells were exposed to palmitic acid (200 μM, Sigma-Aldrich, USA). For Oil Red O staining, AML-12 cells were cultured in 24-well plates and incubated with palmitic acid (200 μM) for 24 h. AML-12 cells were cultured with PPARα agonist WY14643 (5 μM, MedChemExpress, USA) 30 min before NMDA administration. Then the cells were collected 24 h after the NMDA administration. HepG2 cells were maintained in Dulbecco's Modified Eagle's Medium (HyClone, Thermo Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Rockville, USA) containing 100 U/mL penicillin and 100 μg/mL streptomycin. HepG2 cells were cultured in 24-well plates and incubated with NMDA (5 mM) for 24 h.
Wy14643
Manufactured by MedChemExpress
Sourced in United States
WY14643 is a laboratory reagent supplied by MedChemExpress. It is a peroxisome proliferator-activated receptor alpha (PPARα) agonist.
Automatically generated - may contain errors
Lab products found in correlation
Palmitic acid, by MedChemExpress (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Anti mfn2, by Proteintech (1 mentions)
Mdivi 1, by MedChemExpress (1 mentions)
Aicar, by MedChemExpress (1 mentions)
Anti vdac1, by Proteintech (1 mentions)
Compound c, by MedChemExpress (1 mentions)
Anti caspase 3, by Proteintech (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Anti mfn1, by Proteintech (1 mentions)
Anti drp1, by Proteintech (1 mentions)
Anti opa1, by Proteintech (1 mentions)
Anti lamin b, by Proteintech (1 mentions)
Anti γ h2a x ab81299, by Abcam (1 mentions)
Anti ampkα 2532, by Cell Signaling Technology (1 mentions)
P ampkα 50081, by Cell Signaling Technology (1 mentions)
Anti pink1, by Proteintech (1 mentions)
Anti parkin, by Proteintech (1 mentions)
4 protocols using wy14643
1
Palmitic Acid and NMDA Effects on Hepatocytes
2
Mitochondrial Dysfunction and Cell Death
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TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) (1:1,000). Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA) (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. (1:1,000).
3
PPAR-Mediated Transcriptional Regulation Assay
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AML12 cells were transfected with PPRE X3-TK-Luc (200 ng/well), β-gal expression vector (200ng/ well), and pSG5 PPARα (200 ng/well) plus other expression vectors (200 ng/well) as indicated using polyethylenimine (Sigma-Aldrich). Then, after growth for 24 h, cells received treatment of WY14643 (5μmol/ L, MedChemExpress), trametinib (100nmol/L, MedChemExpress), SCH772984 (50 nmol/L, MedChemExpress), or IKK16 (1 μmol/L, MedChemExpress) for another 24 h. Luciferase activity was determined with luciferase reporter assay system (Promega, Madison, WI, USA). β-gal activity, as the control for transfection efficiency, was measured by a kit (Mairybio Biological, Beijing, China). Results were averaged over three biological replicates.
4
Protein Expression Analysis in HepG2 Cells
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HepG2 cells were firstly cultured with GB1 or Wy14643 (HY-16995, MedChemExpress, Monmouth Junction, NJ, USA) in a petri dish (6 cm). Following PBS washing two times, the cells were lysed in lysis buffer to obtain total proteins. Equal amounts of protein were separated by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was subsequently blocked with 5% skim milk, followed by incubation with anti-PPARα (ab61182, Abcam, Shanghai, China), anti-SIRT6 (A3591, ABclonal, Woburn, MA, USA), anti-BCL-2 (T40056, Abmart, Berkeley Heights, NJ, USA), anti-β-actin (RM3002, Beijing Ray antibody biotech, Beijing, China), and anti-BCL-XL (T55050, Abmart, Berkeley Heights, NJ, USA) antibodies. Relative expression of the proteins was normalized to actin protein. The total load of the protein was 60 µg/µL. The dilution ratio of primary antibody was 1:1000, and that of secondary antibody was 1:5000. The type of secondary antibody was Goat anti-Rabbit. Chemiluminescence signals were quantified using a chemiluminescence imaging system (BIO RAD, Hong Kong, China).
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