Topcount plate reader

HEK293T cells stably expressing wild‐type or tyrosine phosphorylation‐deficient HaloTag‐VEGFR2, as well as NFAT‐RE‐luc2P, were grown to 70–80% confluency. Cells were seeded at 25,000 cells per well in white 96‐well plates pre‐coated with poly‐D‐lysine. Following 24 hr at 37%/5% CO₂, cell culture media were replaced with serum‐free DMEM for another 24 hr. Cells were then stimulated with increasing concentrations of VEGF₁₆₅a (R&D Systems) or vehicle (serum‐free DMEM/0.1% BSA). Following stimulation for 5 hr at 37%/5% CO₂, media were replaced with 50 μl per well assay buffer and 50 μl per well ONE‐Glo Luciferase reagent (Promega Corporation, USA). Cells were incubated for 5 min to allow luciferase to react with the added reagent and for the background luminescence to subside, and then luminescence emissions were measured using a TopCount platereader (PerkinElmer, UK). Data were normalised to their respective vehicle (0%) and response of wild‐type HaloTag‐VEGFR2 to 10‐nM VEGF₁₆₅a (100%) per experiment. Data were pooled from five independent experiments with duplicate wells.

VEGFR2 NFAT cells were seeded in a T75 flask at 5 × 10⁶ cells per flask using DMEM +10%FCS and incubated at 37°C in a 5% CO₂/95% air atmosphere for 3 days until the cells were 100% confluent. On the fourth day, cells were washed with PBS and detached using 3 mL Versene® (ETDA 0.02% in PBS). Once cells had detached, 6 mL of DMEM +0.1%BSA was added and the cells were counted using a haemocytometer. Cells were centrifuged at 200× g for 5 min, resuspended in DMEM +0.1%BSA and seeded at a density of 4 × 10⁴ cells per well in 80 μL DMEM +0.1%BSA in white-sided, clear flat-bottomed 96-well plates (Greiner, Stonehouse, UK), which had been coated with 0.01 mg·mL⁻¹ poly-D-lysine in PBS for 30 min and washed with DMEM. Cells were then incubated for 1 h in a humidified 5% CO₂/95% air atmosphere at 37°C. RTKIs or vehicle control were added in 10 μL DMEM +0.1%BSA for 1 h prior to addition of VEGF₁₆₅a or VEGF₁₆₅b in 10 μL DMEM +0.1%BSA and the incubation was continued for a further 5 h (in a humidified 5% CO₂/95% air atmosphere at 37°C). After the 5 h incubation, 100 μL ONE-Glo Luciferase Assay reagent was added to each well and luminescence was measured according to the manufacturer's instructions on a Topcount platereader (Perkin Elmer, Llantrisant, UK).

HEK293T cells stably expressing both wild type VEGFR2 and the Firefly luciferase reporter gene ReLuc2P (Promega Corporation, USA) inserted downstream of the NFAT promoter were used to monitor NFAT-induced gene transcription following VEGFR2 activation (Carter et al., 2015 (link)). On the day of experimentation, cells grown to 95-100% confluency were plated in white-sided 96 well plates (Greiner Bio-One, 655089) at 44,000 cells/well, and incubated for 1 hour in 100μl/well serum free DMEM/0.1% BSA (37°C/5% CO₂). Cells were stimulated in duplicate wells with increasing concentrations of VEGF₁₂₁a-TMR, VEGF₁₆₅b-TMR or equivalent unlabelled VEGF isoforms (synthesised in an identical manner to the fluorescent variant), then incubated for 5 hours at 37°C/5% CO₂. ONE-Glo Luciferase reagent (Promega Corporation, USA) was then added at 100μl/well and luminescence was measured using a TopCount platereader (Perkin Elmer, UK) following a 5 minute delay allowing reagent to react with luciferase and background luminescence to subside.

Specific binding of [³H]BMS-955176 to Gag was demonstrated using a scintillation proximity assay (SPA), which is a radiolabeled binding assay. A total of 0.5 μg of WT VLPs (PBS solution) was mixed with 100 μg of SPA beads (PBS suspension; PVT WGA SPA beads; PerkinElmer) in a total volume of 40 μl per well (96-well white low-binding plate [Corning, Corning, NY]). After a 1-h incubation at room temperature, the volume was increased to 180 μl/well by the addition of binding buffer (100 mM Tris [pH 6.5], 2 mM EDTA, 0.03% Tween 20, 5 mM MgCl₂). A K_d (dissociation constant) determination was made by adding 20 μl of a serial dilution of [³H]BMS-955176 (0.2 to 600 nM) to the VLP mixture. The data were fit to an equation for saturation binding (GraphPad v 5.1). For determination of a K_i value, 11 nM [³H]BMS-955176 was added to the VLP-bead mixtures, to which was added a serial dilution of unlabeled BMS-955176 or BVM. After 4 h of equilibration at room temperature, bound [³H]BMS-955176 was measured using a Top Count plate reader (PerkinElmer). The data were fit to an equation for homologous competition (GraphPad v 5.1).
See the supplemental material for experimental details concerning the following: cytotoxicity assay, off-target activity assays, two-drug combination assays, HIV-1 cell fusion assay, and ultracentrifugation serum binding assay.

Histone methyltransferase assays were performed in 96-well half-area optiplates (PerkinElmer) at room temperature. All concentrations are final unless noted otherwise. Compounds were serially diluted in DMSO and the final DMSO concentration was 1%. A 24 μL reaction mixture contained compound, enzyme, 200 nM of nucleosome or histone substrate, 400 nM ³H-SAM (83.1 Ci/mmol), 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 0.01% Tween-20. The plate was sealed, mixed on a Titramax plate shaker at 1200 rpm for 30 sec, and incubated at room temperature for 60 min. The reaction was quenched by addition of 24 μL mixture containing 5 mg/mL PVT-PEI beads and 300 μM unlabeled SAM in water. The plate was sealed, mixed as previously noted, incubated overnight, and read on a TopCount plate reader (PerkinElmer).