Fig. 3. .

Small molecule validation data. (A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format, fixed, and stained for analysis on the LI-COR Aerius instrument using the Alexa-680 succinimidyl ester as a counter stain for cell number and the phospho-rpS6 antibody followed by an IRDye-800CW secondary antibody for quantitation of rpS6 phosphorylation. The dose-dependent inhibition of rpS6-staining was fit with a standard 4-parameter model giving an IC₅₀ of 85.1 pM (red curve). In a parallel experiment, cells were serum-starved overnight, treated with increasing amounts of rapamycin, stimulated with EGF, fixed, and stained using the Alexa-680 succinimidyl ester and the phospho-ERK antibody followed by an IRDye-800CW secondary antibody for quantitation of ERK phosphorylation (blue curve), showing that rapamycin does not inhibit ERK phosphorylation. Each data point is an average of 4 independent experiments. The raw image from the LI-COR Aerius scanner is shown on the right. (B) A similar dose–response experiment was performed using the PI3-kinase inhibitor, LY294002, showing an IC₅₀ of 3.31 µM for the phospho-S6 assay (red curve) (LY294002 was significantly less active toward the MAPK pathway showing an IC₅₀ > 0.25 mM (blue curve). (C) A dose response using the MEK inhibitor, UO126, was performed showing no inhibition of rpS6-phosphorylation across a range of concentrations (red curve). In a parallel experiment, the activity of the UO126 compound was confirmed, showing a dose-dependent inhibition of ERK phosphorylation in response to EGF stimulation with an IC₅₀ of 0.622 µM (blue curve). (D) The statistical reproducibility of the assay for small molecule screening was determined by calculating a Z′ for the assay comparing 20 ng/mL rapamycin to DMSO control in a 384-well format. The raw data for this assay is shown in the left panel, with the phospho-rpS6 signal (green) overlaid with the Alexa-680 succinimidyl ester cell number stain (red). The quantitation of the raw data is shown in the right panel and was used to calculate a Z′ of 0.80 for this experiment. The asterisks (*) denote untreated wells that were not used in the Z′ determination.