Validation & Assay Performance Summary

GeneBLAzer ® AR-UAS-bla GripTite ™ Cells

Cat. no. K1698

Target Description

The androgen receptor (AR) is a validated drug target of the nuclear receptor superfamily. AR antagonists like flutamide and bicalutamide are used in the treatment of androgen- dependent prostate cancer.

Cell Line Description

GeneBLAzer ® AR-UAS-bla GripTite™ 293 cells contain the ligand-binding domain (LBD) of the rat Androgen receptor (AR) fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer ® UAS-bla GripTite™ 293 cell line. This portion of the rat AR is identical to the human AR in the conserved LBD and differs from the human sequence at five residues in the hinge region. GeneBLAzer ® UAS-bla GripTite™ cells stably express a beta-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS). When an agonist binds to the LBD of the GAL4 (DBD)-AR (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase.

® GeneBLAzer AR-UAS-bla GripTite™ 293 cells are functionally validated for Z’ and EC 50 concentrations of R1881 (Figure 1). In addition, AR-UAS-bla GripTite™ 293 cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Additional testing data using alternate stimuli are also available.

www.invitrogen.com/drugdiscovery 09Nov2010

Validation Summary Primary Agonist Dose Response

Performance of this assay was validated under Figure 1 — AR-UAS-bla GripTite ™ dose response to optimized conditions in a 384-well format using known agonist R1881 LiveBLAzer™-FRET B/G Substrate. 5 1. Primary agonist dose response under optimized conditions (n=6) 4

R1881 EC 50 = 0.4 nM 3 Z’-Factor (EC 100 ) = 0.68 Response Ratio = 3.4 2 Optimum cell no. = 8K cells/well Optimum [DMSO] = up to 0.5% 1

Stimulation Time = 16 hours Response Normalized Max. [Stimulation] = 30 nM 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 2. Alternate agonist dose response log [R1881], M See agonist dose response section AR-UAS -bla GripTite™ 293 cells (8,000 cells/well) were plated 3. Antagonist dose response in a 384-well format and serum starved for 24 hours. Cells See antagonist dose response section were then stimulated with a dilution series of R1881 in the presence of 0.5% DMSO for 16 hours. Cells were subsequently

4. Cell culture and maintenance loaded with LiveBLAzer™-FRET B/G Substrate (1 µM final concentration of CCF4-AM) for 2 hours. Fluorescence emission See Cell Culture and Maintenance Section and values at 460 nm and 530 nm were obtained using a standard Table 1 fluorescence plate reader and date from three individual users were plotted for each replicate against the concentrations of R1881. Assay Testing Summary

5. Assay performance with variable cell Alternate Agonist Dose Response number Figure 2 — AR-UAS-bla GripTite ™ dose response to known agonists R1881 and DHT 6. Assay performance with variable DMSO concentration

7. Assay performance with variable 1.5 R1881 stimulation time DHT

1.0 Dexamethasone 8. Assay performance with variable Hydroxyflutamide substrate loading time 0.5 Emission Ratio 460 nm: 530 nm

0.0 -11 -10 -9 -8 -7 -6

Log [Ligand], M

AR-UAS-bla GripTite ™ cells (8,000 cells/well) were plated the day of the assay in a 384-well format. Cells were stimulated with either R1881, DHT (dihydrotestosterone), dexamethasone, or hydroxyflutamide over the indicated concentration range in the presence of 0.5% DMSO for 16 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate (1 µM final concentration of CCF4-AM) for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the ratios were plotted against the indicated concentrations of the agonists.

www.invitrogen.com/drugdiscovery 09Nov2010 Antagonist Dose Response Cell Culture and Maintenance

Figure 2 — AR-UAS-bla GripTite ™ dose response to known Cells should be maintained at between 5 and 90% antagonists OHF, Spironolactone and Cyproterone Acetate confluency in complete growth media and in a

humidified incubator at 37ºC and 5% CO 2. Split 25 nM DHT cells at least twice a week. Do not allow cells to

0.8 reach confluence. OHF 0.7 Spiro

Cyp Ac 0.6 Tam 0.5

0.4

Ratio Blue:Green 0.3

0.2 -10 -9 -8 -7 -6 -5 log [ligand], M

AR-UAS-bla GripTite ™ cells (8,000 cells/well) were plated the day of the assay in a 384-well black-walled tissue culture assay plate. Cells were treated with OHF (hydroxyflutamide), Spironolactone, Cyproterone Acetate and tamoxifen (as a specificity control) and incubated at 37 degrees C for 45 min., followed by 25 nM DHT (dihydrotestosterone) agonist stimulation for 16 hours in 0.5% DMSO. Cells were then loaded for 120 minutes with LiveBLAzer™- FRET B/G Substrate. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the ratios were plotted against the indicated concentrations of ligand.

Table 1 – Cell Culture and Maintenance

Growth Medium Growth Medium Component Assay Medium Freeze Medium (–) (+) DMEM, w/ GlutaMAX TM 90% 90% — — Opti-MEM — — 90% — Dialyzed FBS 10% 10% 10% — Do not substitute! NEAA 0.1 mM 0.1 mM 0.1 mM — Sodium Pyruvate — — 1 mM — HEPES (pH 7.3) 25 mM 25 mM — — Hygromycin — 80 µg/mL — — Zeocin® — 80 µg/mL — — Penicillin 100 U/mL 100 U/mL 100 U/mL — Streptomycin 100 µg/mL 100 µg/mL 100 µg/mL — Recovery ™ Cell Culture — — — 100% Freezing Medium

www.invitrogen.com/drugdiscovery 09Nov2010 Assay Performance with Variable Cell Assay Performance with variable stimulation Number time

Figure 3 – R1881 dose response with 6, 8, 12 and 15K Figure 5 – AR-UAS-bla GripTite ™ dose response to cells per well R1881 with 5, 8, and 16 hour stimulation times

0.7 AR Assay: Agonist Time Course

0.6 6k 0.5 8k 16h 0.5 12k 8h 0.4 15k 0.4 5h 0.3 0.2 Ratio Blue:Green 0.1 0.3

0.0 Ratios Blue:Green -12 -11 -10 -9 -8 -7 log [R1881], M 0.2 -13 -11 -9 -7 -5 AR-UAS-bla GripTite ™ cells were plated at 6,000, 8,000, log [R1881], M 12,000 or 15,000 cells/well in a 384-well format the day of the assay in 0.5%DMSO. Cells were stimulated with R1881 for 16 AR-UAS-bla GripTite ™ cells (8,000 cells/well) were plated the hours. Cells were then loaded with LiveBLAzer™-FRET B/G day of the assay in a 384-well black-walled tissue culture Substrate (1 µM final concentration of CCF4-AM) for 2 hours. assay plate in 0.5% DMSO. R1881 was then added to the plate Fluorescence emission values at 460 nm and 530 nm for the over the indicated concentration range for 5, 8, and 16 hours various cell numbers were obtained using a standard and then loaded for 2 hours with LiveBLAzer™-FRET B/G fluorescence plate reader and the ratios were plotted against Substrate (1 µM final concentration of CCF4-AM). Fluorescence the indicated concentrations of R1881. emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the ratios were plotted against the indicated concentrations of R1881.

Assay Performance with variable DMSO concentration Assay Performance with variable substrate Figure 4 – AR-UAS-bla GripTite ™ dose response to loading time R1881 with 0, 0.25, 0.5 and 1% DMSO. Figure 6 – AR-UAS-bla GripTite ™ dose response to R1881 with 90, 120 and 150 minute substrate loading 0.6 0% time 0.5 0.25% 0.7 0.5% 0.4 0.6 150 min 1% 0.3 0.5 120 min 0.2 0.4 90 min 0.3 Ratios Blue:Green 0.1 0.2 0.0 Ratio Blue:Green -14 -13 -12 -11 -10 -9 -8 -7 -6 0.1

log [R1881], M 0.0 -14 -13 -12 -11 -10 -9 -8 -7 AR-UAS-bla GripTite ™ cells (8,000 cells/well) were plated the log [R1881], M day of the assay in a 384-well black-walled tissue culture bla assay plate. R1881 was then added to the plate over the AR-UAS- GripTite ™ cells were plated at 8,000 cells/well in a indicated concentration range. DMSO was added to the assay 384-well format the day of the assay in 0.5% DMSO. Cells at concentrations from 0% to 1%. Cells were stimulated for 16 were stimulated with R1881 for 16 hours. Cells were then hrs with agonist and loaded for 2 hours with LiveBLAzer™- loaded with LiveBLAzer™-FRET B/G Substrate (1 µM final FRET B/G Substrate (1 µM final concentration of CCF4-AM). concentration of CCF4-AM) for either 90, 120, or 150 minutes. Fluorescence emission values at 460 nm and 530 nm were Fluorescence emission values at 460 nm and 530 nm for the obtained using a standard fluorescence plate reader and the various loading times were obtained using a standard ratios were plotted for each DMSO concentration against the fluorescence plate reader and the ratios were plotted against indicated concentrations of R1881. the indicated concentrations of R1881.