Spectromax 250

All reagents for the MESG-based microtubules-activated ATPase assay were obtained from Cytoskeleton. Reactions were set up in wells of a 96-well plate (Corning Costar No. 3697) and each well contained 50 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 0.1 mM sodium azide, 20 μM paclitaxel, 0.5 μM MT, 0.5 mM ATP, 0.1 unit purine nucleoside phosphorylase2 (PNP), 0.2 mM MESG reagent, and kinesin proteins in a reaction volume of 200 μl. NaCl (150 mM) was added to the assay conditions for WT and F73A mutant Z601. Reactions were started by the addition of ATP and were read every 10 s at 360 nm for a total of 20 min using a monochromatic spectrophotometer (SpectroMax250, Molecular Devices, San Diego, CA). The assay is based on an absorbance shift (330–360 nm) that occurs when MESG is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate and PNP.

Immulon 4HBX 96-well microtiter plates (Thermo Scientific) were coated with 0.1 mL of anti-RTA mAb (1 µg/ml) diluted in PBS (pH 7.4) and incubated overnight at room temperature. Wells were blocked at 4 °C with block solution [PBS containing 1% (v/v) Tween-20 and 2% (v/v) goat serum]. The concentration of biotinylated RT (biotin-RT) used in the EPICC assay was equivalent to the EC₉₀ for each RT-specific mAb (range = 100–500 ng/ml for PB10/R70, SyH7, IB3, GD12). Biotin-RT at appropriate EC₉₀ concentration was mixed with 1:25 dilutions of sera from naïve and vaccinated rhesus macaques and applied to the mAb-coated plates. The plates were incubated for 1 h at room temperature, washed, then overlaid with streptavidin-HRP (1 μg/ml). The plates were washed again to remove unbound streptavidin-HRP and developed using TMB according to the manufacturer’s instructions (Kirkegaard & Perry Labs, Gaithersburg, MD). Optical densities at 450 nm (OD₄₅₀) for each well were determined using a SpectroMax 250 spectrophotometer (Molecular Devices) equipped with Softmax Pro 7.0 software. Inhibition of RT capture by a given mAb was expressed as a percent (%) reduction derived from the OD₄₅₀ values of wells without (B) and with (C) competitor, as follows: [100-(OD₄₅₀C/OD₄₅₀B)*100].

GI₅₀ (50% growth inhibitory concentration) values were determined by the sulforhodamine B (SRB) staining method [39 (link)]. Cells were seeded in 96-well plates (SJSA-1; 2×10³ cells/well, NGP; 5×10³ cells/well) and allowed to attach for 24 hours before exposure to Nutlin-3 or MI-63 for 72 hours. Nutlin-3 and NU7441/Rucaparib were administered concomitantly. Cells were fixed in Trichloroacetic Acid (10% v/v), stained with SRB (0.4% w/v) and the absorbance measured at 570 nM using a SpectroMax 250 (Molecular Devices, Berkshire, UK) microwell plate scanner. GI₅₀ values were calculated using GraphPad PRISM software (GraphPad Software, Inc., San Diego, CA, USA) and fold-resistance determined. For assessment of cell confluence and proliferation over time, cells were seeded 24 hours before treatment and confluence was measured by IncuCyte^® Zoom (Essen BioSciece) every 6 hours.

Dose and time dependent cell viability assay was carried out with LN-18 cells and G1 cells using MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide) (Sigma Aldrich). 5 × 10³ cells were grown in 96 well plates for 24 h to obtain 80% confluency and then treated using fresh medium with serial concentrations of RAP, TEM, TOR and PP242 for 24 h, 48 h or 72 h time periods. The effect of mTOR inhibitors in “washout” experiments was assessed in G-1 cells treated with RAP (10 μM), TEM (5 μM), TOR (100 nM) or PP-242 (100 nM) for 24 or 48 h. The media containing inhibitor was aspirated and cells were washed with media and replenished with inhibitor-free fresh complete media and the cells were further incubated for 24 h or 48 h respectively. Another set of treated cells was maintained without “washout” for 72 h time period and was regarded as control set. To terminate the experiments, control and test samples were incubated with 0.5 mg/ml of MTT in PBS for 4 h. The formazan crystals formed were dissolved using 10% SDS and absorbance was measured at 570–640 nm using microplate reader (Spectromax 250, Molecular Devices). The percentage of viable cell count was calculated assuming control viable cell count as 100%.