Nutlin 3

Romidepsin (FK228) was obtained from Gloucester Pharmaceuticals (Cambridge, MA, USA). Pre-designed siRNAs for non-targeting Control, Akt1 and Mdm2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nutlin-3 was obtained from R&D Systems (Minneapolis, MN, USA). Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein isothiocyanate-conjugated anti-CXCR2 (FAB331F, 1:1) was from R&D Systems (Minneapolis, MN, USA) and propidium iodide was from Sigma-Aldrich (St. Louis, MO, USA). PCR array for the cell cycle, SYBR^® Green Master Mix, RNeasy Mini Kit and RNase-free DNAse set came from SABiosciences in Qiagen (Frederick, MD, USA). Chemiluminescent detection kits were from GE Healthcare (Piscataway, NJ, USA). Penicillin G/streptomycin, Lipofectamine 2000 and all liquid culture media were acquired from Invitrogen (Grand Island, NY, USA). Mycoalert mycoplasma detection kit was from Lonza (Allendale, NJ, USA). G418-sulfate was purchased from Cayman Chemical (Ann Arbor, MI, USA). Finally, the Luciferase Reporter Assay System was obtained from Promega (Madison, WI, USA).

The LNCaP prostate carcinoma cell line (ATCC, Middlesex, UK) was selected for its ability to internalise ¹³¹I‐MIP‐1095 and to grow spheroids. LNCaP cells were maintained in Roswell Park Memorial Institute medium supplemented with 10% (v/v) hyclone foetal calf serum (Fisher Scientific, Loughborough, UK), 4 mm L‐glutamine, 10 mm HEPES, 2.52 g/l D‐glucose and 1 mm sodium pyruvate. Unless otherwise stated, all reagents used for cell culture were purchased from Life Technologies (Paisley, UK). DSF, Cu and topotecan were purchased from Sigma‐Aldrich (Dorset, UK). Nutlin‐3 was purchased from Biotechne—R&D systems (Oxon, UK). Olaparib, bortezomib and AZD7762 were purchased from Stratech Scientific Ltd (Suffolk, UK). Only, topotecan and Cu were dissolved in aqueous solutions. DSF, Nutlin‐3, Olaparib, bortezomib and AZD7762 were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO used for the treatment of spheroids was 0.1% (v/v). Stock solutions of drugs were prepared at 1000 times the required concentration.