Mouse microglial BV2 cells (Cell Resource Center of the Chinese Academy of Medical Sciences) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Beijing Aoqing Biotechnology Co., Ltd.) and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere with 5% CO2 and the medium was changed every 2 days. Cells were split with 0.125% trypsin when they reached 80% confluence and the passages 2-10 were used for carrying out experiments. For LPS treatment, the cells were treated with 100 ng/ml LPS (cat. no. L2630; Sigma-Aldrich; Merck KGaA) for the indicated times. To perform the hypoxia exposure experiments, BV2 cells seeded in 60-mm dishes (8x105 cells/dish) were put in the cell culture chamber with 1 or 3% oxygen (O2) at 37˚C for the indicated times. For the FG-4592 (Selleck Chemicals) treatment, cells were treated with 10 µM FG-4592 for 6, 12 and 24 h at 37˚C. For the celecoxib (Selleck Chemicals) treatment, cells were pretreated with 10 and 20 µM celecoxib for 1 h at 37˚C.
Fg 4592
Manufactured by Selleck Chemicals
Sourced in United States
FG-4592 is a laboratory equipment product designed for use in scientific research and analysis. It serves as a device for the measurement and monitoring of various parameters, such as temperature, pressure, or other physical characteristics, within a controlled laboratory environment. The core function of FG-4592 is to provide accurate and reliable data collection to support scientific investigations and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (3 mentions)
Anti vhl, by Cell Signaling Technology (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Anti actb, by ABclonal (2 mentions)
Anti hif 1α, by Cell Signaling Technology (2 mentions)
Cocl2, by Merck Group (2 mentions)
Anti ha, by Fortrea (2 mentions)
Roxadustat, by Selleck Chemicals (2 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Cyclohexamide, by Abcam (1 mentions)
Sirnas, by Thermo Fisher Scientific (1 mentions)
4 thiouridine, by Merck Group (1 mentions)
Anti m6a polyclonal antibody, by Synaptic Systems (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
Hif 1β, by Novus Biologicals (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Hif 1α, by BD (1 mentions)
Celecoxib, by Selleck Chemicals (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
21 protocols using fg 4592
1
Microglial Cell Culture and Treatments
2
FG-4592 Pretreatment in Rats
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Animals were randomly divided into the following two groups of 36 animals each. Group A (the experimental (FG-4592) group) rats received oral FG-4592 (Selleck Chemicals, Houston, TX, USA) as a single dose of 60 mg/kg every other day and Group B (the control group) rats received only tap water during the experiment. The dose of FG-4592 used was based on a patent [19 ]. The first drug solution was administered to the animals 7 days before the surgical procedure, and FG-4592 was administered using a mouth gag and feeding tube. The final administration was performed 2 h before the surgical procedure.
3
SH-SY5Y Neuroblastoma Cell Culture
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SH-SY5Y neuroblastoma cells were cultured in Dulbecco’s modified eagle’s medium (DMEM, Hyclone) containing 10% fetal calf serum (Hyclone, Logan City, UT, United States) and grown in a CO2 incubator maintained at atmospheric oxygen levels and 5% CO2. Dissolved MPP+ (Sigma Aldrich, St. Louis, MO, United States) in phosphate buffered saline (PBS) to a storage concentration of 125 mM and MPP+ was added to the culture medium to achieve the final concentration. FG-4592 (Selleckchem, Houston, TX, United States) stocks were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 50 mM. Right before each experiment, a stock of FG-4592 was added to the culture medium to achieve the final concentration at 50 μM. Thus 0.1% of DMSO was present in the final culture. They were both stored at -20°C. At MPP+ and FG-4592 co-treatment group, FG-4592 was added 6 h before MPP+.
SH-SY5Y cells were transiently transfected with siRNA-HIF-1α (against HIF-1α) or siRNA-NC (as a negative control) using lipofectamine2000 (Invitrogen, Carlsbad, CA, United States). siRNAs were purchased from Biotend Co. Ltd. (Shanghai, China), the sequence of siRNA is as followed: F: UGAUACCAACAGUAACCAA; R: UUGGUUACUGUUGGUAUCA.
SH-SY5Y cells were transiently transfected with siRNA-HIF-1α (against HIF-1α) or siRNA-NC (as a negative control) using lipofectamine2000 (Invitrogen, Carlsbad, CA, United States). siRNAs were purchased from Biotend Co. Ltd. (Shanghai, China), the sequence of siRNA is as followed: F: UGAUACCAACAGUAACCAA; R: UUGGUUACUGUUGGUAUCA.
4
Zinc and PHI FG4592 Modulate VSMC Calcification
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Human aortic smooth muscle cells (VSMCs) were purchased from Cell Applications (San Diego, CA, United States, Cat.: 354-05a) and Lonza (Allendale, NJ, United States Cat.: CC-2571). Zinc dose experiments were performed on cells purchased from Cell Applications, while all the other experiments were performed on VSMCs purchased from Lonza. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 1000 mg/L glucose supplemented with 10% fetal bovine serum (Life Technologies, Vienna, Austria), 100 U/ml penicillin, 100 μg/ml streptomycin, and neomycin referring to as growth medium (GM). Cells were grown to confluence and used from passages 5 to 7. Media were changed every 2 days. To induce phosphate mediated calcification, GM was supplemented with inorganic phosphate (3 mmol/L referring to as calcification medium) in a form of Na2HPO4/NaH2PO4 (pH 7.4) in the presence or absence of zinc (15–30 μmol/L of ZnCl2 × 6H2O) for 10 days. To investigate the effect of PHI FG4592 on VSMC calcification, cells were treated with FG4592 (Selleckchem, Houston, United States) in various concentrations (5 and 20 μmol/L) in calcification medium in the presence or absence of zinc (30 μmol/L) for 3, 6 or 10 days.
5
Comprehensive Cellular Protein Regulation
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FG-4592 was obtained from either Selleckchem or MedChemExpress. Cyclohexamide was purchased from Abcam. siRNAs were obtained from Thermo Fisher Scientific, MG132 and 4-thiouridine, and N-ethylmaleimide were purchased from Sigma-Aldrich. Primary antibodies used in this study are ALKBH5 (Atlas Antibodies), β Actin (Sigma), HIF-1α (BD Bioscience), HIF-2α (NOVUS), HIF-1β (NOVUS), NDRG1 (CST), HBc (DAKO), anti-m6A polyclonal antibody (Synaptic Systems). HRP-conjugated secondary antibodies were purchased from DAKO.
6
Cell Viability Assay with FG-4592
7
Synthesis of HIF Stabilizers
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8
Pharmacological Modulation of Proteostasis
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DMSO was used as vehicle control. The proteasome inhibitor MG132 was obtained from Merck/Millipore and used at the final concentration of 20 μM for 3 h. PHD inhibitors IOX2 and FG-4592 were purchased from Selleckchem. VHL inhibitors VH032, VH298, and nonbinding epimer cisVH298 were synthesized by a group member of our laboratory (13 (link), 14 (link)). Compounds were added to cells for indicated length of time. Chloroquine (Merck) and bafilomycin A1 (Selleckchem) were added to cells for 3 h at 50 μM and 50 nM, respectively.
9
Reagents for Hypoxia Signaling Pathway
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Anti-SMYD3 (#ab199361) antibody was purchased from Abcam. Antibodies including anti-HIF1α (#36169), anti-VHL (#68547), anti-Histone H3 (#4499), anti-HIF2α (#7096), anti-ARNT (#5537), and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology. Anti-ACTB (#AC026) antibody was purchased from ABclonal. Anti-HA (#901515) antibody was purchased from Covance. Anti-Myc (#SC-40) and anti-GAPDH (#SC-477242) antibodies were purchased from Santa Cruz Biotechnology. Anti-α-tubulin (#62204), Alexa Fluor 488 goat anti-rabbit IgG (#A11008), Alexa Fluor 594 goat anti-mouse IgG (#A11005), CM-H2DCFDA (#C6827), and MitoSOX Red (#M36008) were purchased from Thermo Fisher Scientific. CoCl2 (#C8661) and deferoxamine mesylate salt (#D9533) were purchased from Sigma. FG4592 (#S1007) and PX478 (#S7612) were purchased from Selleck. Cycloheximide (#HY-12320) was purchased from MCE.
10
FG4592 Modulates Cell Apoptosis and Viral Infection
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For FG4592 (Selleck, #S1007) treatment of cells, H1299 or THP-1 cells were treated with FG4592 (0–100 μM) for 24 h, followed by cell apoptosis analysis; or the cells were treated with FG4592 (0–100 μM) for 6 h, followed by virus infection and then qPCR or flow cytometry analysis.
Mice treated with FG4592 were approximately 6–8 weeks old mice (male). FG4592 was prepared as a 10 mg/ml solution in vehicle (5% DMSO, 40% PEG 300, 5% Tween 80 and 50% ddH2O). Mice were treated with 10 mg/kg FG-4592 or vehicle alone by oral gavage twice daily. Mice were then infected with VSV (5 × 107 PFU per mouse) by intraperitoneal injection followed by the indicated analysis.
For FG4592 treatment of zebrafish, wild-type zebrafish larvae (3 days post-fertilization, dpf) or Tg(ifnφ1:mCherry) zebrafish larvae (8 dpf) in egg water were treated with FG4592 (10 μM) for 24 h, followed by SVCV infection and indicated analysis.
Mice treated with FG4592 were approximately 6–8 weeks old mice (male). FG4592 was prepared as a 10 mg/ml solution in vehicle (5% DMSO, 40% PEG 300, 5% Tween 80 and 50% ddH2O). Mice were treated with 10 mg/kg FG-4592 or vehicle alone by oral gavage twice daily. Mice were then infected with VSV (5 × 107 PFU per mouse) by intraperitoneal injection followed by the indicated analysis.
For FG4592 treatment of zebrafish, wild-type zebrafish larvae (3 days post-fertilization, dpf) or Tg(ifnφ1:mCherry) zebrafish larvae (8 dpf) in egg water were treated with FG4592 (10 μM) for 24 h, followed by SVCV infection and indicated analysis.
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