Pipeline pilot platform

To monitor proliferation of reporter cell lines in the presence of library compounds, 15 μL of DME-L medium [20 (link)] containing 5 mM glucose and 10% heat inactivated fetal bovine serum was dispensed into each well of 384-well microplates (black polystyrene, clear bottom, tissue culture treated, Corning) with a Matrix Wellmate liquid dispenser (Thermo Scientific). Stock compounds dissolved in dimethyl sulfoxide (DMSO) were pin-transferred (V&P Scientific) into the microplate to the desired final concentration using an automated robot arm. 15 μL of the reporter cell line, e.g., Δlmxgt1-3[pPfHT], at 2 x 10⁶/mL was added per well with the Wellmate dispenser. Microplates were incubated (Liconic) at 28°C and 5% CO₂ for 72 h. After incubation, 10 μL of the reading solution (5X SYBR Green prepared from a commercial 100X stock, SIGMA, 5% Triton-X in PBS) was added per well. Each plate was shaken at 1000 rpm for one min, incubated further at room temperature for 20 min, and then fluorescence was read (excitation 485 nm, emission 535 nm) with the Envision plate reader (PerkinElmer). All data processing and visualization, as well as chemical similarity and substructure analysis, was performed using custom programs written in the Pipeline Pilot platform (Accelrys, v.7.0.1) and the R program [21 ].

To measure uptake of glucose in a medium throughput format, 100 μL/well of PBS was added to 96-well filter plates (Millipore) with the Wellmate dispenser. Control wells included either 0 mM (positive control) or 20 mM (negative control) of the competitive inhibitor fructose in PBS. Compounds were added with the Biomek FX automated system. 90 μl of a cell suspension (1.1 x 10⁸ cells/mL) was added to each well with the Wellmate dispenser and left at room temperature for 5 minutes before adding 10 μL of the substrate (4 mM [³H] D-glucose at 50 μCi/mL) to provide a final glucose concentration of 200 μM. After a 5 min incubation, the uptake reaction was stopped by adding 50 μL/well of 4% formaldehyde, followed by incubation for ~5 min. Cells were filtered and washed with a vacuum manifold (Millipore). Plates were dried overnight, 100 μL of scintillation fluid were added, and plates were then sealed and read on a TopCount NXT HTS from PerkinElmer. Data quality and analysis was performed using the GUItars program [22 (link)] and sigmoidal curve fitting with Pipeline Pilot platform (Accelrys, v. 7.0.1).