Iodine 125

Iodination with iodine-125 was done in pre-coated iodination tubes (Pierce). 100 mg of transferrin was dispersed in 200 μL of PBS solution and added to the pre-coated iodination tubes. In a separate eppendorf, 1 μL of HCl (0.2 M), 2.5 μL of phosphate buffer (0.5 M, pH = 8), 10 μL of potassium iodide solution (1 mg/ml) was prepared. 3 mCi of iodine-125 (Perkin Elmer) was added into the tubes and the previous solution was then mixed in the iodination tubes. After 15 min of reaction at room temperature the solution was purified via PD10 column pre equilibrated with 20 mL of PBS solution. The purity was assessed via iTLC, and ¹²⁵I-Tf was always > 98% pure.
PC3 and 22Rv1 cells were counted and plated at fixed cell concentrations between treatment arms. Cells were treated with vehicle or the bromodomain inhibitors iBET-151 or JQ1 (1 μM) for 48 hours, whereupon they were washed and incubated with 10 μCi ¹²⁵I-Tf for 30 min at 37° C. After washing twice with PBS, the cell associated activity was harvested in 1M NaOH (aq.). The cell associated activity was expressed as a % of total activity to which the cells were exposed. This value was further normalized to cell number to correct for treatment-induced changes in cell viability.

All data presented in this study are available upon request. All materials and chemicals were purchased from commercial vendors and used without further processing/purification. DU145, 22RV1, C4-2B, and PC3 cell lines were obtained from American Type Tissue Collection (ATCC) and cultured according to manufacturer’s recommendations. Cellular identity was authenticated by visually inspecting morphology and probing for signature expression markers on immunoblot. Mycoplasma contamination was tested within the first two passages after thawing cryostocks with the MycoAlert kit (Lonza). All cells were studied between passages 5 to 25. The monoclonal antibody 4A06 was expressed and purified in the IgG1 format as previously described (13 ). p-SCN-Bn-Deferoxamine (B-705) and p-SCN-Bn-DOTA (B-205) were purchased from Macrocyclics (Plano, TX). ⁸⁹Zr-oxalate was obtained from 3D Imaging, LLC (Maumelle, AR). ¹⁷⁷LuCl₃ was obtained from Oak Ridge National Laboratory. Iodine-125 was obtained from Perkin Elmer. ⁶⁸Ga-PSMA 11 was prepared by the radiopharmacy at UCSF according to previously reported protocol (17 (link)). LTL xenograft samples were acquired form Living Tumor Laboratory (Vancouver, BC) and the LuCaP xenograft series was provided by Dr. Eva Corey at University of Washington.