Unc1062

Prostatosphere formation assays were performed using a slight modification of previously described techniques [39 (link), 40 (link)]. Cells were plated in DMEM F-12 (11320-033, Life Technologies) containing 10 ng/mL bFGF (233-FB/CF, R&D Systems, Minneapolis, MN), 20 ng/mL EGF (236-EG, R&D Systems), 5 mg/mL insulin (3435, R&D Systems), and 0.4% (v/v) bovine serum albumin (BSA) (5217, R&D Systems) supplemented with 1% (v/v) knockout serum replacement (10828-028, Life Technologies) at 500-3,000 cells per well in 6-well ultra low attachment plates. In some case, the cultures were treated with the Mer inhibitor UNC1062 (AOB4488, AOBIOUS, Gloucester, MA). Prostatosphere formation (cell clusters of 10 cells or greater) was observed at 7–10 days under a light microscopy.

Proliferation was assessed using Thiazolyl Blue Tetrazolium Bromide (MTT) Assay. 2500 HN cells overexpressing MERTK or GFP were plated in 100 μl medium in 96-Well plates (Corning, Corning, NY, USA). 500 μg/ml MTT (Sigma-Aldrich, Munich, Germany) dissolved in phosphate buffered saline (PBS) (Gibco® Life technologies, Darmstadt, Germany) was added at different time points. Four hours after treatment with MTT 100 μl solubilization buffer (40 % vol/vol Dimethylformamide (Alfa Aesar, Karlsruhe, Germany), 2 % vol/vol glacial acetic (Merck, Darmstadt, Germany), 16 % wt/vol sodium dodecyl sulfate (Applichem, Darmstadt, Germany), pH 4.7) was added and absorbance at 595 nm was measured the next day. For inhibition experiments 5000 Detroit 562 or 2500 HN cells were plated in 50 μl medium before adding different amounts of UNC1062 (AOBIOUS, Gloucester, MA, USA) in 50 μl medium the next day. Knockdown cells were induced for 72 hours with 1 μg/ml doxycycline before plating 5000 cells in 100 μl medium. MTT was added after different time points as described above.
Each experiment was performed in triplicates and repeated at least three times.