Validation & Assay Performance Summary

GeneBLAzer ® RXR beta DA Cells & Assay Kit

GeneBLAzer ® RXR beta-UAS-bla HEK 293T Cells

Cat. no. K1405, K1694

Target Description

The Retinoid X -beta (RXR beta) is a nuclear and can function as a ligand inducible capable of acting as a co-repressor and/or co- activator for expression. Nuclear receptors contain a series of conserved domains or regions. These domains/regions include a variable NH 2-domain (A/B region), a conserved DNA-binding domain (DBD or region C), a linker region (region D), a ligand binding domain (LBD or region E), and in some receptors a variable COOH-terminal (region F) (1). RXR beta belongs to the family of retinoid x receptors, one of two retinoic receptor families ( receptors and retinoid x receptors). RXR beta is one of three members of the RXR family which consists of RXR alpha, RXR beta, and RXR gamma (11). The A/B and D regions of RXR beta are involved in dictating the cell dependent transcriptional response (6). RXR receptors are able to form both homo- and heterodimers (3). RXR receptors have been reported to form heterodimers with TRs (thyroid hormone receptors), RXRs retinoic acid receptors), VDR (), PPARs (peroxisome proliferator activated receptors), LXRs (liver x receptors), and FXR (). These heterodimers can be classified as permissive and nonpermissive heterodimers (3). Addition of an RXR agonist, such as 9-cis-retinoic acid, can result in transcriptional activity of permissive heterodimers while activation of nonpermissive heterodimers occurs independent of the RXR agonist (3). RXR agonist LG100268 activates the transcriptional response RXR:PPARγ and RXR:LXR heterodimers alone and synergistically with PPARγ and LXR agonists, but does not activate RXR:RXR and RXR:TR heterodimers whose activity is dependent upon RXR and TR agonists (10). RXR beta is widely expressed in almost every tissue. The generation of RXR beta null mice resulted in approximately 50% of the mice to die before birth and those that survived appear normal, except for male sterility (reviewed in 11). The endogenous ligands for RXR beta include 9-cis-retinoic acid (4,6), phytanic acid (12), and (7). Synthetic agonists for RXR beta have been termed rexinoids and includes LG100268 (10).

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Cell Line Description

GeneBLAzer ® RXR beta DA (Division Arrested) cells and RXR beta-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human beta (RXR beta) fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer ® UAS-bla HEK 293T cell line. GeneBLAzer ® UAS-bla HEK 293T 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)-RXR beta (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase.

DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both RXR beta DA cells and RXR beta-

UAS-bla HEK 293T cells are functionally validated for Z’ and EC 50 concentrations of 9-cis retinoic acid (9-cis-RA) (Figure 1). In addition, RXR beta-UAS-bla HEK 293T cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, and stimulation time.

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Validation Summary 11. Dose Response with additional agonists in the presence of Performance of this assay was validated under RXRα antagonist RO41-5253 optimized conditions in 384-well format using LiveBLAzer™-FRET B/G Substrate. 12. Effect of pre-incubating cells on assay performance 1. Primary agonist dose response under optimized conditions (n=3)

DA Dividing 9-cis retinoic acid EC 50 4.8 nM 12 nM Z’-Factor (EC 100 ) 0.79 0.74

Response Ratio = 4-5 Optimum cell no. = 10K cells/well Optimum [DMSO] = up to 1% Stimulation Time = 16 hours Max. [Stimulation] = 10 µM

2. Alternate agonist dose response

All-trans-retinoic acid EC 50 = 22.6 nM Phytanic acid EC 50 = 6.9 µM Docosahexaenoic acid EC 50 = 10 µM

3. Antagonist dose response See antagonist dose response section

4. Cell culture and maintenance See Cell Culture and Maintenance Section and Table 1

Assay Testing Summary

5. Assay performance with variable cell number

6. Assay performance with variable stimulation time

7. Assay performance with variable substrate loading time

8. Assay performance with variable DMSO concentration

9. Dose Response with additional nuclear receptor agonists

10. Assay performance with variable assay plate types

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Figure 1 —9-cis-retinoic acid dose response under Figure 2 —9-cis-retinoic acid, all-trans-retinoic acid, optimized conditions phytanic acid, and docosahexaenoic acid agonist dose Response

1.75 2.5 Dividing Cells 9-cis-retinoic acid 1.50 DA Cells 2.0 All-trans-retinoic acid 1.25 Phytanic acid 1.00 1.5 DHA 0.75 1.0 0.50 460/530 ratio 460/530 460/530 Ratio 460/530 0.5 0.25

0.00 0.0 -2 -1 0 1 2 3 4 5 -11 -10 -9 -8 -7 -6 -5 -4 log [9-cis-retinoic acid] nM Log M [agonists]

Dividing RXR beta-UAS-bla HEK 293T cells were serum starved for RXR beta-UAS-bla HEK 293T cells (10,000 cells/well) were 24 hrs prior to the assay, while division arrest cells were thawed serum starved for 24 hrs prior to the assay, and then plated and assayed directly. The day of the assay, cells were plated in a the day of the assay in a 384-well format. Cells were 384-well format (10,000 cells/well) and stimulated with 9-cis- stimulated with either 9-cis-retinoic acid (Biomol #GR101), all- retinoic acid (Biomol #GR101) over the indicated concentration trans-retinoic acid (Sigma #R2500), phytanic acid (Sigma cat# range in the presence of 1.0% DMSO for 16 hours. Cells were P4060) or docosahexaenoic acid (DHA) (Sigma #D2534) over then loaded with LiveBLAzer™-FRET B/G Substrate (1 µM final the indicated concentration range in the presence of 1.0% concentration of CCF4-AM) for 90 minutes. Fluorescence emission DMSO for 16 hours. Cells were then loaded with LiveBLAzer™- values at 460 nm and 530 nm were obtained using a standard FRET B/G Substrate (1 µM final concentration of CCF4-AM) for fluorescence plate reader in bottom-read mode, and the 90 minutes. Fluorescence emission values at 460 nm and 530 background-corrected ratios were plotted against the indicated nm were obtained using a standard fluorescence plate reader concentrations of 9-cis-retinoic acid (n= 16 for each data point). in bottom-read mode, and the background-corrected ratios were plotted against the indicated concentrations of the agonists (n= 8 for each data point).

Antagonist Dose Response

There are currently no commercially available antagonists for RXR beta

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Table 1 – Cell Culture and Maintenance

Component Growth Medium (–) Growth Medium (+) Assay Medium Freeze Medium

90% 90% — — DMEM, w/ GlutaMAX TM Phenol Red free DMEM — — 98% —

Dialyzed FBS 10% 10% — — Do not substitute! Charcoal/Dextran FBS — — 2%

0.1 mM 0.1 mM 0.1 mM — NEAA

25 mM 25 mM — — HEPES (pH 7.3)

— 100 µg/mL — — Hygromycin B

— 100 µg/mL — — Zeocin TM

100 U/mL 100 U/mL 100 U/mL — Penicillin

100 µg/mL 100 µg/mL 100 µg/mL — Streptomycin Sodium Pyruvate — — 1 mM

Recovery ™ Cell Culture — — — 100% Freezing Medium

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Figure 4— 9-cis-retinoic acid dose response with 2.5, Figure 6 – 9-cis-retinoic acid dose response with 1, 1.5, 5.0, 10, and 20K cells/well 2, 3, and 4 hour loading times

5.5 10.0 60 min 5.0 20K cells/w ell 4.5 10K cells/w ell 90 min 4.0 5.0K cells/w ell 7.5 120 min 3.5 2.5K cells/w ell 180 min 3.0 2.5 5.0 240 min 2.0 1.5

Response Ratio Response 2.5

1.0 Ratio Response 0.5 0.0 0.0 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 Log M [9-cis-retinoic acid] Log M [9-cis-retinoic acid] RXR beta-UAS-bla HEK 293T cells were plated at 2500, 5,000, RXR beta-UAS-bla HEK 293T cells were plated at 10,000 10,000 or 20,000 cells/well in a 384-well format and serum cells/well in a tissue culture treated 384-well plate format the starved for 24 hours prior to the assay. The cells were then day of the assay after a 24 hour starvation in assay media. stimulated with a serial dilution of 9-cis-retinoic acid (Biomol Cells were stimulated with 9-cis-retinoic acid (Biomol #GR101) #GR101) in the presence of 0.5% DMSO for 18 hours. Cells in the presence of 0.5% DMSO for 16 hours. Cells were then were then loaded with LiveBLAzer™-FRET B/G Substrate (1 µM loaded with LiveBLAzer™-FRET B/G Substrate (1 µM final final concentration of CCF4-AM) for 90 min. Fluorescence concentration of CCF4-AM) for either 1, 1.5, 2, 3, or 4 hours. emission values at 460 nm and 530 nm for the various cell Fluorescence emission values at 460 nm and 530 nm for the numbers were obtained using a standard fluorescence plate various loading times were obtained using a standard reader in bottom-read mode, and the background-corrected fluorescence plate reader in bottom-read mode, and the Response Ratios were plotted against the indicated background-corrected Response Ratios were plotted against concentrations of 9-cis-retinoic acid (n=8 for each data point). the indicated concentrations of 9-cis-retinoic acid (n=8 for

each data point). Assay performance with Variable Stimulation

Time Assay Performance with variable DMSO

concentration Figure 5 – 9-cis-retinoic acid dose response with 6, 16, and 24 hour stimulation times Figure 7 – 9-cis-retinoic acid dose response with 0, 0.1, 3.5 0.5 and 1% DMSO. 6hrs

3.0 16 hrs 3.5 1.0% DMSO 24 hrs 2.5 3.0 0.5% DMSO 2.0 2.5 0.1% DMSO 0.0% DMSO 1.5 2.0 1.0 1.5 Response Ratio Response 0.5 1.0 Response Ratio Response 0.5 0.0 -11 -10 -9 -8 -7 -6 -5 0.0 Log M [9-cis-retinoic acid] -12 -11 -10 -9 -8 -7 -6 -5 Log M [9-cis-retinoic acid] RXR beta-UAS-bla HEK 293T cells (20,000 cells/well) were plated in a 384-well black-walled tissue culture assay plate and RXR beta-UAS-bla HEK 293T cells (20,000 cells/well) were serum starved for 24 hours prior to the assay. The cells were plated the day of the assay in a 384-well black-walled tissue then stimulated with a serial dilution of 9-cis-retinoic acid culture assay plate after a 24 hour starvation in assay media. (Biomol #GR101) in the presence of 0.5% DMSO for 6, 16, 9-cis-retinoic acid (Biomol #GR101) was then added to the and 24 hours. Cells were then loaded for 90 minutes with plate over the indicated concentration range. DMSO was LiveBLAzer™-FRET B/G Substrate(1 µM final concentration of added to the assay at concentrations from 0% to 1%. Cells CCF4-AM). Fluorescence emission values at 460 nm and 530 were stimulated for 16 hrs with agonist and loaded for 90 nm were obtained using a standard fluorescence plate reader minutes with LiveBLAzer™-FRET B/G Substrate (1 µM final in bottom-read mode, and the background-corrected Response concentration of CCF4-AM). Fluorescence emission values at Ratios were plotted against the concentrations of 9-cis-retinoic 460 nm and 530 nm were obtained using a standard acid (n=8 for each data point). fluorescence plate reader in bottom-read mode, and the background-corrected Response Ratios were plotted for each DMSO concentration against the indicated concentrations of 9- cis-retinoic acid (n=8 for each data point).

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Figure 8a – Dose response of additional nuclear Figure 9a. – 9-cis-retinoic acid dose response with receptor agonists. 20K, 10K, 5K, and 2.5K cells/well in a poly-d-lysine assay plate. 3.5 9-cis-RA 3.5 3.0 T3 20K cells/w ell 3.0 2.5 T0901317 10K cells/w ell 2.0 2.5 5.0K cells/w ell 2.5K cells/w ell 1.5 2.0 1.0 1.5 Response Ratio Response 0.5 1.0 Response Ratio Response 0.0 0.5 -12 -11 -10 -9 -8 -7 -6 -5 -4 0.0 Log M [agonist] -12 -11 -10 -9 -8 -7 -6 -5 Log M [9-cis-retinoic acid] RXR beta-UAS-bla HEK 293T cells (10,000 cells/well) were plated the day of the assay in a 384-well black-walled Figure 9b. – 9-cis-retinoic acid dose response with CellBind plate. Cells were stimulated with a serial dilution 20K, 10K, 5K, and 2.5K cells/well in a CellBind assay series of 9-cis-retinoic acid (Biomol cat# GR101), plate. TO901317 (Calbiochem cat# 575310), or T3 (thyroid hormone 3,5,3’-triiodothyronine) (Calbiochem cat# 6 642511) in the presence of 0.5% DMSO for 16 hours at 20K cells/w ell 37ºC. Cells were loaded for 90 minutes with 5 10K cells/w ell LiveBLAzer™-FRET B/G Substrate (1 µM final 5.0K cells/w ell 4 concentration of CCF4-AM). Fluorescence emission values 2.5K cells/w ell at 460 nm and 530 nm were obtained using a standard 3 fluorescence plate reader in bottom-read mode, and the background-corrected Response Ratios were plotted 2 against the indicated concentrations of agonists (n=8 for Ratio Response 1 each data point). 0 -12 -11 -10 -9 -8 -7 -6 -5 Figure 8b – Dose response of additional nuclear Log M [9-cis-retinoic acid] receptor agonists. Figure 9c. – 9-cis-retinoic acid dose response with 4 20K, 10K, 5K, and 2.5K cells/well in a tissue culture 9-cis-retinoic acid treated assay plate. all-trans-retinoic acid 3 AM580 8 20K cells/w ell TTNPB 7 10K cells/w ell 2 GW7647 6 5.0K cells/w ell

5 2.5K cells/w ell 1 Response Ratio Response 4 3 0 2

-12 -11 -10 -9 -8 -7 -6 -5 -4 Ratio Response Log M [agonist] 1 0 RXR beta-UAS-bla HEK 293T cells (10,000 cells/well) were -12 -11 -10 -9 -8 -7 -6 -5 plated the day of the assay in a 384-well black-walled Log M [9-cis-retinoic acid] CellBind plate. Cells were stimulated with a serial dilution series of 9-cis-retinoic acid (Biomol cat# GR101), all- RXR beta-UAS-bla HEK 293T cells were plated the day of trans-retinoic acid (Sigma cat# R2625), TTNPB (Sigma the assay at 20,000, 10,000, 5,000, or 2,500 cells/well in cat# T3757), GW7674 (Sigma cat# G6793), or AM580 either a 384-well black-walled (a) poly-d-lysine, (b) (Sigma cat# A8843) in the presence of 0.5% DMSO for 16 CellBind, or (c) tissue culture treated assay plate. Cells hours at 37ºC. Cells were loaded for 90 minutes with were stimulated with a serial dilution series of 9-cis- LiveBLAzer™-FRET B/G Substrate (1 µM final retinoic acid (Biomol cat# GR101) in the presence of 1.0% concentration of CCF4-AM). Fluorescence emission values DMSO for 16 hours at 37ºC. Cells were loaded for 90 at 460 nm and 530 nm were obtained using a standard minutes with LiveBLAzer™-FRET B/G Substrate (1 µM final fluorescence plate reader in bottom-read mode, and the concentration of CCF4-AM). Fluorescence emission values background-corrected Response Ratios were plotted at 460 nm and 530 nm were obtained using a standard against the indicated concentrations of agonists (n=8 for fluorescence plate reader in bottom-read mode, and the each data point). background-corrected Response Ratios were plotted against the indicated concentrations of agonists (n=8 for each data point.

Tel: 800 955 6288 ● E-mail: [email protected] ● www.invitrogen.com/drugdiscovery 03012006 Dose Response with additional nuclear receptor agonists in the presence and RXR beta-UAS-bla HEK 293T cells (10,000 cells/well) were plated the day of the assay in a 384-well black-walled absence of RXRα antagonist RO41-5253 CellBind plate. Cells were treated with and without 10 µM RO41-5253 (Biomol cat# G110) in the presence of 1% Figure 10a. – Dose response of 9-cis-retinoic acid in DMSO for 30 minutes at 37 °C. Cells were then stimulated the presence or absence of RO41-5253 with a serial dilution series of 9-cis-retinoic acid (Biomol cat# GR101), all-trans-retinoic acid (Sigma cat# R2625), 11 TTNPB (Sigma cat# T3757), or AM580 (Sigma cat# 10 A8843) for 16 hours at 37ºC. Cells were loaded for 90 9 minutes with LiveBLAzer™-FRET B/G Substrate (1 µM final 8 concentration of CCF4-AM). Fluorescence emission values 7 6 at 460 nm and 530 nm were obtained using a standard 5 fluorescence plate reader in bottom-read mode, and the 4 background-corrected Response Ratios were plotted 3 9-cis-RA against the indicated concentrations of agonists (n=8 for Response Ratio Response 2 9-cis-RA w / 10 uM RO41-5253 each data point). 1 0 -10 -9 -8 -7 -6 -5 -4 Log M [9-cis-retinoic acid]

Figure 10b. – Dose response of all-trans-retinoic Effect of pre-incubating cells on assay acid in the presence or absence of RO41-5253 performance All-trans-RA 9 8 All-transRA w / 10 uM RO41-5253 Figure 11. – Dose response of 9-cis-retinoic acid 7 upon seeding of cells or after >3 hr pre-incubation

6 5 4 10.0 3

Response Ratio Response 2 7.5 1 0 -10 -9 -8 -7 -6 -5 -4 5.0 agonist addition at seeding Log M [all-trans-retinoic acid] agonist addition after inc 2.5 Figure 10c. – Dose response of AM580 in the Ratio Response presence or absence of RO41-5253 0.0 2.5 AM580 -12 -11 -10 -9 -8 -7 -6 -5 -4

2.0 AM580 w ith 10 uM RO41-5253 Log M [9-cis-retinoic acid]

1.5

1.0 RXR beta-UAS-bla HEK 293T cells (10,000 cells/well) were Response Ratio Response 0.5 plated the day of the assay in a 384-well black-walled 0.0 CellBind plate. After either a 0 hour or >3 hour pre- -10 -9 -8 -7 -6 -5 -4 incubation at 37 °C, the cells were stimulated with a serial [M] dilution series of 9-cis-retinoic acid (Biomol cat# GR101) in the presence of 1.0% DMSO for 16 hours at 37 °C. Cells Figure 10d. – Dose response of TTNPB in the were loaded for 90 minutes with LiveBLAzer™-FRET B/G presence or absence of RO41-5253 Substrate (1 µM final concentration of CCF4-AM). 3.5 Fluorescence emission values at 460 nm and 530 nm were TTNPB obtained using a standard fluorescence plate reader in 3.0 TTNPB w ith 10 uM RO41-5253 bottom-read mode, and the background-corrected 2.5 Response Ratios were plotted against the indicated 2.0 concentrations of agonists (n=8 for each data point). 1.5 1.0 Response Ratio Response 0.5 0.0 -10 -9 -8 -7 -6 -5 [M]

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References

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3. Mangelsdorf, D.J., et.al. (1995) The RXR heterodimer and Orphan Receptors . Cell , 83 , 841- 850.

4. Allenby, G., et.al. (1993) Retinoic acid receptors and retinoic x receptors: interactions with endogenous retinoic acids . Proc. Nat. Acad. Sci. , 90 , 30-34.

5. Levin, A.A., et.al (1992) 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXRα . Nature , 355 , 359-361.

6. Hallenbeck, P.L., et.al. (1996) Differential 9-cis-retinoic acid dependent transcriptional activation by murine retinoid x receptor (RXRα) and RXR beta . Jour. Biol. Chem ., 271(18) , 10503-10507.

7. Mata de Urquiza, A., et.al. (2000) Docosahexaenoic acid, a ligand for the retinoic x receptor in mouse brain . Science , 290 , 2140-2144.

8. Kizaki, M., et.al. (1993) Effects of novel retinoic acid compound, 9-cis retinoic acid on proliferation, differentiation, and expression of α and retinoid x receptor-α RNA by HL60 cells . Blood , 82(12) , 3592-3599.

9. Liu, H., et.al. (2004) Retinoid X receptor α (RXRα) Helix 12 plays an inhibitory role in the recruitment of the P160 co-activators by unliganded RXRα/retinoic acid receptor α heterodimers . Jour. Biol. Chem ., 279(43) , 45208-45218.

10. Mukherjee, R., et.al. (1997) Sensitization of diabetic and obese mice to insulin by retinoid x receptor agonists. Nature , 386 , 407-410.

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