Tgf β alk inhibitor a83 01

FACS-isolated basal and luminal cells from WT and Pb-Csf1 mice were cultured in Corning® Matrigel® Basement Membrane Matrix (Corning, Tewksbury, MA) with advanced DMEM/F12 supplemented with B27 (Life technologies, Grand Island, NY), 10 mM HEPES, glutamax (Life technologies, Grand Island, NY), penicillin/streptomycin, and the following growth factors: EGF 50 ng/ml (Peprotech, Rocky Hill, NJ), 500 ng/ml recombinant R-spondin1 (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant Noggin (Peprotech, Rocky Hill, NJ), 200 nM of TGF-β/Alk inhibitor A83-01 (Tocris, Ellisville, MO), and 10 μm Y-27632 (Tocris, Ellisville, MO). Dihydrotestosterone (DHT) (Sigma, St. Louis, MO) was added to a final concentration of 1 nM.

The organoid culture was performed as described previously (Karthaus et al., 2014 (link)). Briefly, dissociated prostate cells from 8-12 week-old C57B1/6 mice were cultured in DMEM/F12 supplemented with B27 (Life technologies, Grand Island, NY), 10 mM HEPES, Glutamax (Life technologies, Grand Island, NY), Penicillin/Streptomycin, and the following growth factors: EGF 50 ng/ml (Peprotech, Rocky Hill, NJ), 500 ng/ml recombinant R-spondin1 (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant Noggin (Peprotech, Rocky Hill, NJ), 200 μM TGF-β/Alk inhibitor A83-01 (Tocris, Ellisville, MO), and 10 μM Y-27632 (Tocris, Ellisville, MO). Dihydrotestosterone (Sigma, St. Louis, MO) was added at 1 nM final concentration. Cells were resuspended in growth factor reduced matrigel (Corning, Corning, NY) and plated in 96-well plates.

Murine prostates were divided into three lobe pairs; AP, DLP and VP. Lobes were enzymatically digested with collagenase type II (Gibco) and subsequently with TrypLE (Gibco). Cells were seeded in growth factor reduced Matrigel (Corning) and overlayed with medium containing the growth factors: EGF 5–50 ng/ml (Peprotech), R-spondin1 conditioned medium or 500 ng/ml recombinant R-spondin1 (Peprotech), Noggin conditioned medium or 100 ng/ml recombinant Noggin (Peprotech) and the 200 nM TGF-β/Alk inhibitor A83-01 (Tocris). Dihydrotestosterone (DHT) (Sigma) was added at 0.1–1 nM. Human prostate samples were obtained from patients undergoing radical prostatectomy according to guidelines from the UMC Utrecht. Prostate tissue was enzymatically digested with collagenase type II and subsequently with TrypLE. Cells were seeded in growth factor reduced Matrigel and cultured in medium containing growth factors as above, with the addition of 10 ng/ml FGF10 (Peprotech), 5 ng/ml FGF2 (Peprotech), 1 µM Prostaglandin E² (Tocris), 10 µM SB202190 (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich) and DHT 0.1–1 nM. For detailed summary of culture medium composition see supplementary methods.