Bms 777607

Three-dimensional growth assays were performed as described with the substitution of agarose to prevent cell adhesion [13] (link). Briefly, 2 × 10⁴ cells were plated on top of 1.0% agarose in 6-well plates in media supplemented with cosmic calf serum (Complete, Thermofisher Scientific) or CSS (Midsci). Cells were left untreated or treated with DMSO (vehicle), Bay-11-7085 (Enzo Life Sciences, 1 μM), Casodex (Selleck Chemicals, 10 μM), or BMS-777607 (Selleck Chemicals, 5 μM) daily. After 10 days, images of spheres were taken using a Zeiss Axiovert S100TV inverted microscope (Carl Zeiss Microscopy), and spheres >25 μm in diameter were counted using ImageJ software. The 25-μm threshold was established based on the average sphere size obtained for the control cells. For 2D growth, 2.5 × 10⁴ cells were plated on 12-well plates and counted every 24 hours. Myc-CaP Ctrl or Myc-CaP Ron OE cells were grown in 2D and treated with DMSO (vehicle), LiCl (Sigma, 10 mM, 4 hours), or Bay-11-7085 (Bay 11, 5 μM, 4 hours) when cells were ~70% confluent prior to fractionation.

Mice were maintained under specific pathogen-free conditions and experimental protocols approved by the University of Cincinnati IACUC. For murine cell injections, 1.0 × 10⁶ cells were injected subcutaneously into the flanks of 6- to 8-week-old male FVB mice as described [33] (link), [49] (link). Tumor growth was measured via calipers, and volume was determined by the formula 0.5 × length × width²[50] (link). Surgical castration was performed as described when tumors reached 1000 mm³[33] (link), [49] (link). Precastrated animals were surgically castrated at 6 weeks of age and allowed to recover for 10 days before injection of cells, as described previously [51] (link). For in vivo kinase inhibitor studies, precastrated mice were treated with 50 mg/kg/day BMS-777607 (Selleck Chemicals) or methocellulose (vehicle) via oral gavage once tumors reached 100 mm³. For human LNCaP and LNCaP RON OE cells, 5 × 10⁶ cells were injected subcutaneously into athymic nude mice (NCr-Foxn1^nu) from Charles Rivers; mice were castrated when tumors reached 500 mm³.

Mice were maintained under specific pathogen‐free conditions and experimental protocols approved by the University of Cincinnati IACUC. For prostate cell injections, 1.0 × 10⁶ cells were injected subcutaneously into the flanks of 6−8‐week‐old male FVB mice as described.¹⁹, ²⁰ Tumor growth was measured via calipers and volume was determined by the formula 0.5 × Length × Width².²¹ Surgical castration was performed as described when tumors reached 1000 mm³.¹⁹, ²⁰ For in vivo kinase inhibitor studies, mice were treated with 50 mg/kg/day BMS‐777607 (Selleck Chemicals) or methylcellulose (vehicle) via oral gavage. Mice treated with clodrosome (Encapsula Nanosciences CLD8909) for macrophage depletion were injected with 200 µl intraperitoneally every 3 days. Hi‐Myc mice (FVB‐Tg(ARR2/Pbsn‐MYC)7Key/Nci, Strain # 01XK8) were obtained through the mouse repository at the National Cancer Institute and crossed with ARR₂Pb‐RON strain B mice which were described previously.¹², ²² Mice were euthanized and tissues were collected for analysis at 30 weeks of age.