Table 1.
AR-TIF2 PPIB HCS Assay Protocol in U-2 OS Cells
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Harvest and centrifuge U-2 OS cells | 5 min, 500 g | Aspirate medium, wash with PBS, trypsinize cells, add the McCoy's 5A medium+10% FBS, and centrifuge |
| 2 | Viable U-2 OS cell count | Viable cell count | Resuspend cells in the culture medium and count the number of trypan blue excluding viable cells in a hemocytometer |
| 3 | Coinfect U-2 OS cell with the TIF2-GFP and AR-RFP adenovirus biosensors | 5 μL rAVs per 106 cells | Incubate rAVs and U-2 OS cells in 1.0 mL culture medium for 1 h at 37°C, 5% CO2, and 95% humidity with periodic inversion (every 10 min) |
| 4 | Adjust U-2 OS cells to the required density and seed into 384-well assay plate | 40 μL of 6.25×104 cells/mL, 2,500 cells per well | Seed assay plates with 2,500 cells per well and incubate overnight at 37°C, 5% CO2, and 95% humidity |
| 5 | Transfer library compounds/DMSO to control wells | 5 μL | 20 μM final concentration in well, 0.2% DMSO |
| 6 | Incubate assay plates | 1 or 3 h | At 37°C, 5% CO2, and 95% humidity |
| 7 | Add DHT to compound treated and Max controls, media to Min controls | 5 μL | 20 nM DHT final in well of compound treated and Max controls, media to Min controls |
| 8 | Incubate assay plates | 30 min | At 37°C, 5% CO2, and 95% humidity |
| 9 | Fix cells | 50 μL | 7.4% formaldehyde containing 2 μg/mL Hoechest 33342 in Ca2+- and Mg2+-free PBS prewarmed to 37°C |
| 10 | Incubate plates | 10–30 min | Ambient temperature |
| 11 | Aspirate fixative and wash 2× with PBS | 85 μL | Aspirate fixative and wash twice with 85 μL Ca2+ and Mg2+ free PBS, 50 μL PBS in well |
| 12 | Seal plates | 1× | Sealed with adhesive aluminum plate seals |
| 13 | Acquire images | 20×, 0.4 NA objective | Images of the Hoechst (Ch1), TIF2-GFP (Ch2), and AR-RFP (Ch3) were sequentially acquired on the ImageXpress Ultra platform using the 405, 488, and 561 nm excitation laser lines, a Quad filter cube set, and individual PMTs for each channel |
| 14 | Image analysis assay readout | Average inner intensity AR-RFP in TIF2-GFP-positive nucleoli | Images were analyzed using the translocation enhanced image analysis module using the average inner intensity parameter to quantify the AR-RFP within TIF2-GFP-positive nucleoli |
Step Notes
1. U-2 OS cells maintained in the McCoy's 5A medium with 2 mM l-glutamine supplemented with 10% FBS and 100 U/mL penicillin and streptomycin in a humidified incubator at 37°C, 5% CO2, and 95% humidity. Cell monolayers (<70% confluent) were washed 1× with PBS and then exposed to trypsin-EDTA until they detach from the surface of the tissue culture flasks. Cells pelleted at 500 g for 5 min in the Sorvall ST 16 Centrifuge with a TX-400 Rotor.
2. Aspirate medium, resuspend pelleted cells in tissue culture medium+FBS, and count the number of trypan blue excluding viable cells in a hemocytometer.
3. U-2 OS cells were coinfected with the TIF2-GFP and AR-RFP adenovirus expression constructs by incubating cells with the manufacturer's recommended volume of virus, typically 5 μL/106 cells, in 1.0 mL culture medium for 1 h at 37°C, 5% CO2, and 95% humidity with periodic inversion (every 10 min) to maintain cells in suspension.
4. U-2 OS cells coinfected with the rAV biosensors were seeded into 384-well black-walled clear-bottom plates, Greiner Bio-One Catalogue No. 781956, BioTek Microflo (BioTek, Winooski, VT), at 2,500 cells per well and incubated for 24 h at 37°C, 5% CO2, and 95% humidity in the McCoy's 5A medium with 2 mM l-glutamine supplemented with 10% FBS and 100 U/mL penicillin and streptomycin.
5. Twenty micromolars of compounds added to wells in columns 3–22 using a Bravo (Agilent Technologies, Inc., Santa Clara, CA) or an Evolution P3 (PerkinElmer, Waltham, MA) automated liquid handler outfitted with a 384-well transfer head.
6. Incubate treated coinfected U-2 OS cells 1–3 h at 37°C, 5% CO2, and 95% humidity.
7. DHT (20 nM final in well) added to Max controls and compound wells, media to Min control wells using a Bravo (Agilent Technologies, Inc.) or an Evolution P3 (PerkinElmer) automated liquid handler outfitted with a 384-well transfer head.
8. Incubate treated coinfected U-2 OS cells±DHT 30 min at 37°C, 5% CO2, and 95% humidity.
9. Aspiration of media and fixative addition automated on BioTek ELx405 (BioTek) plate washer.
10. Ten to thirty minutes incubation at ambient temperature to fix cells and stain nuclei with Hoechst.
11. Aspiration of fixative and PBS wash steps automated on BioTek ELx405 (BioTek) plate washer.
12. Plates sealed with adhesive aluminum plate seals using the Abgene Seal-IT 100 plate sealer (Abgene, Rochester, NY).
13. Plates loaded into the ImageXpress Ultra confocal HCS platform (Molecular Devices LLC, Sunnyvale, CA) for scanning using a Catalyst robotic plate handler (Thermo Fisher Scientific, Waltham, MA).
14. Images analyzed using the translocation enhanced image analysis module of MetaXpress (Molecular Devices LLC).
AR, androgen receptor; DHT, dihydrotestosterone; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GFP, green fluorescent protein; HCS, high-content screening; PBS, phosphate-buffered saline; PMT, photomultiplier tube; PPIB, protein–protein interaction biosensor; rAV, recombinant adenovirus; RFP, red fluorescent protein; TIF2, transcriptional intermediary factor 2.