FDA approved compound screening library (978 compounds, 10 mM solution in DMSO, L1300) was purchased from Selleckchem. For the validation of the selected compounds, we separately ordered levosimendan (Cat. #S2446, SelleckChem; Cat. #L5545, Sigma), spironolactone (Cat. #S4054, SelleckChem; Cat. #S3378, Sigma), 9-aminoacridine (Cat. #92817, Sigma), and mycophenolic acid (Cat. #S2487, SelleckChem; Cat. #M3536, Sigma). Recombinant human TNFα (Cat. #BD 554618) was purchased from BD Bioscience. 3′-Azido-3′-deoxythymidine (AZT, Cat. #A2169) was purchased from Sigma. Raltegravir (RAL, Cat. #S2005) were purchased from SelleckChem. 3-Methyladenine (3-MA, Cat. S2767) was purchased from SelleckChem. Anti-human CD3 antibody (Cat. #16-0037-85) and antihuman CD28 antibody (Cat. #16-0289-85) were purchased from eBioscience.
L1300
Manufactured by Selleck Chemicals
Sourced in United States
The L1300 is a laboratory vacuum pump designed for general use in various scientific applications. It provides a reliable and consistent vacuum performance to support various laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
P3xflag cmv 14 expression vector, by Merck Group (2 mentions)
Plateloc, by Agilent Technologies (2 mentions)
3 azido 3 deoxythymidine azt, by Merck Group (1 mentions)
9 aminoacridine, by Merck Group (1 mentions)
Spironolactone, by Selleck Chemicals (1 mentions)
Mycophenolic acid, by Selleck Chemicals (1 mentions)
Raltegravir ral, by Selleck Chemicals (1 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (1 mentions)
E1980, by Promega (1 mentions)
Maestro, by Schrödinger (1 mentions)
Cellomics arrayscan vti, by Thermo Fisher Scientific (1 mentions)
Bioactive compound library, by Targetmol (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
12 protocols using l1300
1
Screening of FDA-Approved Compounds Library
2
High-throughput Cytotoxicity Screening Assay
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Cells were plated in 384 well plates (Greiner Bio-One, 781091) at a density of 2,500 cells/well. Cells were allowed to attach overnight, were washed in media to remove dead or unattached ECs, and the following day cells were treated with the FDA-approved small compound library comprised of 1,080 small molecules (L1300, Selleck Chemicals, Houston Texas) on a robotic platform (Microlab STAR system, Hamilton Robotics, Reno, NV). Cells were treated with 25 µL of each compound at 2x (to a final concentration of 10 µM), were incubated at 37 C for 4 hours, followed by removal of the medium and drug, and incubation with 2.5% complement-intact human serum (Sigma-Aldrich, St. Louis, MO) for another 4 hours. PrestoBlue assay solution (Life Technologies, Carlsbad, CA) was prepared and added to each well at a final concentration of 1x (from 10x stock). Plates were incubated at 37 C for 1 hr, followed by a 30 min incubation at room temperature in the dark, and cell death was quantified using an automated plate reader (EnVision, Perkin Elmer, Waltham, MA). For the initial screen, all samples were evaluated in duplicate.
3
Screening FDA-approved Drugs for PRL-3 Inhibition
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The library of FDA-approved drugs was from Selleck (L1300). For further testing, single drugs were purchased as listed in Supplemental Table 4 . Thienopyridone was generously provided by Dr. Zhong-Yin Zhang (Purdue University). PRL-3 overexpressing construct was made by cloning full length PRL-3 cDNA into p3XFLAG-CMV-14 expression vector (Sigma, Cat # E7908).
4
HEK 293T Luciferase Assay for Compound Screening
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HEK 293 T cells were co-transfected with a plasmid expressing Firefly luciferase and a plasmid expressing Renilla luciferase. After 24 h, FDA-approved compounds (20 μM, L1300, Selleckchem), containing 2,570 compounds, were added separately to the culture medium. The cells were lysed using lysis solution, and the luciferase activity was examined by adding luciferase assay substrate (E1980, Promega). The results were shown as log2-fold change normalized to control.
5
Screening FDA-Approved Compound Library
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An FDA-approved compound library containing in total 1’172 FDA-approved drugs (L1300, Selleck Chemicals) was purchased as 100μl 10mM DMSO stock and sealed under argon gas to prevent deterioration using a thermal sealer (Agilent PlateLoc). Hit molecules that were selected after screening the FDA-approved compound library as described below were re-ordered through SelleckChemicals and provided as 10mM stock solutions in DMSO or manually dissolved to 10mM in DMSO.
6
Molecular Docking of FDA-Approved Drugs
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Molecular docking of FDA-approved drugs (L1300, Selleck Inc., Pittsburgh, PA, USA) against NDM-1 was performed in three stages using Glide (Schrodinger, LLC, New York, NY, USA), as described previously [54 (link),55 (link)]. In the first stage, HTVS was conducted on the FDA-approved drugs to get a rough estimation of the docking energies of ligands towards the target protein. In the second stage, the ligands displaying good docking energies in HTVS were further subjected to SP docking, wherein the protein–ligand complexes were optimized by post-docking minimization. In the third stage, the ligands shortlisted after SP docking were subjected to more stringent XP docking, wherein the scaling factor and partial charge cutoff were set to 0.80 and 0.15, respectively [55 (link)]. Finally, the ligands showing the best docking energies towards NDM-1 were subjected to post-docking analysis in Maestro (Schrodinger, LLC, New York, NY, USA).
7
High-Throughput Adipocyte Screening
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For high-throughput chemical screenings, the Ucp1-2A-GFP preadipocytes were differentiated in 96-well plates. At day 8 after differentiation, cells were treated with chemicals for 24 h and analyzed for GFP and DAPI signals using High Content Screening (HCS) (Cellomics ArrayScan VTI, Thermo Fisher Scientific). The ratio of GFP /DAPI signal of each well was used to evaluate the effect of each chemical. The chemical library was purchased from Selleck (L1300) and applied in a final concentration of 10 μM of each chemical in screening. Cells treated with DMSO and isoproterenol were used as controls.
8
Screening FDA-Approved Compound Library
9
Screening FDA-approved drugs for lipid reduction
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To identify potential effective drugs from the FDA-approved drug library (#L1300, Selleckchem, USA), we employed the human hepatocyte cell line L02. The FDA-approved drugs library was procured from Selleck. 10,000 hepatocytes were seeded per well in a 96-well plate. The day following plating, we exposed the cells to 0.5 mM/1 M PA/OA for 18 h while simultaneously administering the drugs. The drugs were administered at a concentration of 20 μM. Bodipy staining was employed to assess lipid accumulation, and the fluorescence intensity was then quantified using a high-content machine.
10
Comprehensive FDA-approved Drug Library
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The library of FDA-approved drugs (Selleck, L1300) was generously provided by Dr.
Vivek Rangnekar at the University of Kentucky. For further testing, additional drugs were purchased as listed in Supplemental Table 3. Thienopyridone was generously provided by Dr.
Zhong-Yin Zhang (Purdue University). PRL-3 over expressing construct was made by cloning full length PRL-3 cDNA into p3XFLAG-CMV-14 expression vector (Sigma, E7908)
Vivek Rangnekar at the University of Kentucky. For further testing, additional drugs were purchased as listed in Supplemental Table 3. Thienopyridone was generously provided by Dr.
Zhong-Yin Zhang (Purdue University). PRL-3 over expressing construct was made by cloning full length PRL-3 cDNA into p3XFLAG-CMV-14 expression vector (Sigma, E7908)
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