HEK293T (ATCC CRL03216) were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 50 units ml−1 of penicillin, streptomycin, and glutamine. Cells were plated 20,000 cells/well in a 96-well flat bottom cell culture plate and co-transfected with 100 ng pG5-UAS and 25 ng pCMV-Gal4-PPARγ (human residues 185–477, isoform 1 numbering, containing the hinge and LBD) along with pACT empty vector (Promega) expressing the VP16 transactivation domain only, pACT with VP16 fused to the mouse NCoR receptor interaction domain (RID, residues 1828–2471), or pAct with VP16 fused to the NCoR RID with each critical residue of the ID2 motif (LEDIIRKAL) mutated to threonine (TEDTTRKAT). Transfection solutions were prepared in Opti-MEM with Mirus Bio TransIT-LT1 transfection reagent. After 24 h incubation at 37 °C in a 5% CO2 incubator, DMSO or T0070907 was added at a final concentration of 0.01% or 10 μM, respectively. After another 24 hr incubation, britelite plus (PerkinElmer) was added to each well, mixed, then transferred to a white-bottom 384-well plate and read on a BioTek Synergy Neo multimode plate reader. Data were plotted and analyzed using GraphPad Prism software.
Synergy neo multi mode plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy Neo Multi-Mode Plate Reader is a high-performance, multi-mode microplate reader designed for a wide range of applications. It can measure absorbance, fluorescence, and luminescence in a single instrument.
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Lab products found in correlation
Britelite plus, by PerkinElmer (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Black low volume 384 well plates, by Greiner (2 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
Kaleidagraph, by Synergy Software (1 mentions)
Dulbecco s minimal essential medium, by Thermo Fisher Scientific (1 mentions)
White 384 well cell culture plates, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
X tremegene 9, by Roche (1 mentions)
Glucose 6 phosphate (g6p), by Merck Group (1 mentions)
Facsdiva v8, by BD (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
C11 bodipy, by BD (1 mentions)
10 protocols using synergy neo multi mode plate reader
1
HEK293T Transfection and Reporter Assay
2
Quantifying 6PGD Activity in Glioma Cells
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6PGD activity was measured at room temperature. 6PGD was immunoprecipitated from the lysates of glioma cells and subjected to 6PGD enzymatic activity assays in the reaction buffer containing 100 mM Tris (PH 7.4), 2 mM 6-phosphogluconate, 10 mM MgCl2, 0.1 mM NADP+. The change in absorbance at 340 nm owing to increase of NADPH was measured using BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, USA).
3
TR-FRET Assay for REV-ERB Ligand Binding
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The TR-FRET assay was performed in black low-volume 384-well plates (Greiner). Each well contained 4 nM 6xHistag-REV-ERBα LBD (human; residues 281–614) or 6xHistag-REV-ERBβ LBD (human; residues 212–579) protein expressed in and purified from Escherichia coli using nickel affinity and size exclusion chromatography; 1 nM LanthaScreen Elite Tb-anti-HIS Antibody; and 400 nM FITC-labeled peptide derived from the SMRT corepressor containing a N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C terminus for stability in TR-FRET buffer (20 mm potassium phosphate, pH 7.4, 50 mm potassium chloride, 1 mm dithiothreitol, and 0.005% Tween-20). Ligand stocks were prepared via serial dilution in DMSO, added to wells in triplicate, and plates were incubated at 4°C for 2 h and read using BioTek Synergy Neo multimode plate reader. The Tb donor was excited at 340 nm; its fluorescence emission was monitored at 495 nm, and the acceptor FITC emission was measured at 520 nm; and the TR-FRET ratio was calculated as the signal at 520 nm divided by the signal at 495 nm. Data were plotted using GraphPad Prism as TR-FRET ratio versus ligand concentration and fit to sigmoidal dose response curve equation.
4
Fluorescence Polarization Binding Assay
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Fluorescence polarization was measured using a BioTek Synergy Neo Multi-Mode plate reader (BioTek) with excitation at 485 nm and detection at 528 nm. For competitive binding experiments, the wild-type CHMP4C peptide (residues 216–233) was synthesized with a nonnative cysteine at the N terminus (CQRAEEEDDDIKQLAAWAT) and was labeled with Oregon Green 488 (Life Technologies/Molecular Probes 06,034) following the manufacturer’s instructions. The labeled peptide was quantitated by the absorbance of Oregon Green 488 at 491 nm (extinction coefficient 83,000 cm⋅M−1 in 50 mM potassium phosphate, pH 9). Different concentrations (as determined by absorbance at 280 nm) of unlabeled N-terminally acetylated CHMP4C peptides were titrated against a CHMP4CA232-ALIX Bro1-V complex created by mixing 5 μM ALIX Bro1-V and 0.5 nM fluorescently labeled CHMP4CA232 peptide in binding buffer [20 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM β-mercaptoethanol, 0.01% Tween-20, and 0.2 mg/mL BSA]. IC50s were calculated from binding curves using KaleidaGraph (Synergy Software) and were converted to Ki values (55 (link)). Competitive binding curves were measured independently seven or more times for each peptide and are expressed as mean ± SD. Ki values are reported. All peptides were synthesized by the University of Utah Peptide Synthesis Core Facility and were verified by mass spectrometry.
5
TR-FRET Assay for REV-ERB Ligand Binding
6
Quantifying PPARγ Transcriptional Activity
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HEK293T (ATCC CRL03216) were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 50 units ml−1 of penicillin, streptomycin, and glutamine. Cells were grown to 90% confluency in T-75 flasks; from this, 2 million cells were seeded in a 10-cm cell culture dish for transfection using X-tremegene 9 (Roche) and Opti-MEM (Gibco) with full-length human PPARγ (isoform 2) expression plasmid (4 μg), and a luciferase reporter plasmid containing the three copies of the PPAR-binding DNA response element (PPRE) sequence (3xPPRE-luciferase) (4 μg). After an 18-h incubation, cells were transferred to white 384-well cell culture plates (Thermo Fisher Scientific) at 10,000 cells/well in 20 μL total volume/well. After a 4 h incubation, cells were treated in quadruplicate with 20 μL of either vehicle control (1.5% DMSO in DMEM media), twofold serial dilution of TZDs for dose response experiments, or 5 μM ligand. After a final 18-h incubation, cells were harvested with 20 μL Britelite Plus (PerkinElmer), and luminescence was measured on a BioTek Synergy Neo multimode plate reader. Data were plotted in GraphPad Prism as luminescence vs. ligand concentration and fit to a sigmoidal dose response curve.
7
Measurement of G6PD Activity in Organoids
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G6PD activity was measured at room temperature. G6PD was immunoprecipitated from the lysates of organoids and subjected to G6PD enzymatic activity assays in the reaction buffer containing 42 mM Tris (PH 7.5), 2.66mM Glucose-6-phosphate (Sigma, G7879-500mg), 40mM MgCl2, 0.66 mM β-NADP. The change in absorbance at 340 nM owing to increase of NADPH was measured using BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, USA).
8
Directional Kinetics of PGK1 Enzyme
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PGK1 WT and PGK1 variants were overexpressed in HaEpi cells individually and PGK1-His were pulled down for the PGK1-catalyzed reaction to investigate the directionality of the PGK1 reaction. PGK1 activity was measured at room temperature in kinetic mode for 5 min by coupling with GAPDH. The absorbance at 340 nm was measured based on changes in NADH concentrations with a BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, USA). PGK1 was isolated from H. armigera cell lysates or fat body and subjected to a PGK1-catalyzed reaction of 3-PG production assays in a reaction buffer containing 5 mM KH2PO4 (pH 7.0), 1 mM glyceraldehyde 3-phosphate (3-GAP), 0.3 mM β-nicotinamide adenine dinucleotide (β-NAD), 0.2 mM ADP, 5 mM MgSO4, 100 mM glycine, and 5 ng/mL GAPDH.
9
Measuring Cellular Oxidative Stress Levels
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Cellular ROS levels were measured using an ROS detection kit (BioVision, Waltham, MA; #K936-100), as previously described48 (link): 50,000 cells were plated in wells and collected, and fluorescence intensity at Ex/Em = 495/529 nm was quantified using a BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, Santa Clara, CA). A representative plot of three biological replicates is shown.
For lipid Peroxidation determination, cells were seeded in DMEM and washed twice in HBSS and treated with Oxaliplatin (Normal-dose, 5 µg ml−1; low-dose, 1 µg ml−1) in DMEM for the indicated time, then incubated in DMEM containing 2 mM BODIPY 581/591 C11 (Invitrogen, D3861) for 30 min at 37 °C. For imaging, slides were excited using the 488 and 565 nm laser and fluorescence measured from 505 to 550 nm and above 580 nm using Zeiss LSM880 microscope. Upon oxidation of the polyunsaturated butadienyl portion of the dye, there is a shift of the fluorescent emission peak from 590 nm to 510 nm, remaining lipophilic and thus reflecting lipid peroxidation. Further examination was performed through a BD laser analyzer using PE-Texas Red (PE-TR) filter (measuring non-oxidized BODIPY-C11) and fluorescein isothiocyanate (FITC) (measuring oxidized BODIPY-C11). A minimum of 10,000 cells were collected using BD FACS Diva v8.0 in each condition and analyzed using FlowJo v10.7 (Bioscience).
For lipid Peroxidation determination, cells were seeded in DMEM and washed twice in HBSS and treated with Oxaliplatin (Normal-dose, 5 µg ml−1; low-dose, 1 µg ml−1) in DMEM for the indicated time, then incubated in DMEM containing 2 mM BODIPY 581/591 C11 (Invitrogen, D3861) for 30 min at 37 °C. For imaging, slides were excited using the 488 and 565 nm laser and fluorescence measured from 505 to 550 nm and above 580 nm using Zeiss LSM880 microscope. Upon oxidation of the polyunsaturated butadienyl portion of the dye, there is a shift of the fluorescent emission peak from 590 nm to 510 nm, remaining lipophilic and thus reflecting lipid peroxidation. Further examination was performed through a BD laser analyzer using PE-Texas Red (PE-TR) filter (measuring non-oxidized BODIPY-C11) and fluorescein isothiocyanate (FITC) (measuring oxidized BODIPY-C11). A minimum of 10,000 cells were collected using BD FACS Diva v8.0 in each condition and analyzed using FlowJo v10.7 (Bioscience).
10
Enzymatic Activity Assay of LOXL3
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To analyze the activity regulation of LOXL3 and of the DHODH-K344 peptide ALEK344IRAGAS, and an oxidation assay was performed as previously described4 (link),51 (link). Briefly, we purified His-tagged LOXL3 from Escherichia coli or FLAG-tagged LOXL3 from mammalian cells, then concentrated the LOXL3 protein to 200 µg ml−1. Activity assays were performed using peptides at 10 mg ml−1. The enzymatic reaction was initiated by adding a substrate mixture containing 50 mM sodium borate (pH 8.2), 100 mM N-acetyl-3,7-dihydroxyphenoxazine, and 20 mM 1,5-diaminopentane. The production of hydrogen peroxide by LOXL3 results in fluorescent resorufin production, which can be measured by excitation at 540 nm and emission at 580 nm. The fluorescence reaction was measured every 5 min for 1.5 h at 37 °C using a BioTek Synergy Neo Multi-Mode Plate Reader (BioTek). Each representative bar data point was derived from three independent replicates.
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