The cultures were maintained in a humidified 5% CO2 environment at 37°C. The medium was changed twice a week and the cells were trypsinized and subcultivated once a week. Somatostatin and Octreotide (Sigma) were prepared as described previously [24]. The cells were treated with 1 nM somatostatin

and 1 nM Octreotide for different periods of time (0, 1 h, 12 h, 24 h, 72 h), as described by Brevini [25]. Controls were untreated cells. RNA extraction and RT-PCR XAF1 mRNA was detected using reverse transcription PCR (RT-PCR). Total cellular RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufactures’ instruction. cDNA was synthesized using random primers (N6) and M-MLV reverse transcriptase. PCR was performed by using

XAF1 -specific primers as follows: forward: 5′-ATG GAA GGA GAC TTC TCG GT-3′; reverse: 5′-TTG CTG AGC Selleck MI-503 TGC ATG TCC AG-3′ and the conditions were: denaturation at 94°C for 5 min, followed by 34 cycles of 94°C 30 s, 60°C 30 s, 72°C 45 s, and then a final cycle of 10 min at 72°C. Amplification products (290 bps) were electrophoresed onto 1.5% agarose gels and visualized by 0.5% ethidium bromide staining. The results of electrophoresis were analyzed by the Gel Image System Fluor Chem TM 9900 (Alpha Innotech). Western blot analysis Cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% NP-40 and 5 mM EGTA, 50 mM sodium flu-oride, 60 mM β-glycerol-phosphate, 0.5 mM sodium-vanadate, 0.1 mM PMSF, 10 μg/ml PD184352 (CI-1040) aprotinin and 10 μg/ml leupeptin. Protein concentration was determined using the BCA protein assay kit (Pierce Bio-technology, Inc., USA). Protein ACP-196 samples (40 μg) were subjected to a 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were first incubated with 5% nonfat milk in Tris-buffered saline (TBS). After washing three times in 0.1% Tween 20-TBS (TBST), the membranes were incubated with primary antibody (goat anti-human XAF1, 1:600;

Santa Cruz Biotecnology) and β-actin (rabbit anti-actin antibody R-22, 1:1000; Santa Cruz Biotecnology) separately at 4°C overnight, followed with the corresponding secondary antibodies separately (1:2500) for 1.5 h at room temperature and the antibody-bound proteins were detected by the ECL system (Amersham Biosciences, Little Chalfont Buckinghamshire, UK). Results Expression of XAF1 mRNA and protein in prostate cell lines The expression of XAF1 was detected at mRNA and protein levels with RT-PCR and Western blot. As shown in Figure 1, RT-PCR using cDNA primers specific for a segment of the human XAF1 mRNA provided a product of the expected size in four prostate cell lines. It showed lower expression of XAF1 mRNA in prostate cancer cells LNCaP, DU145 and PC3 compared with that in RWPE-1 cells which displayed the strongest expression of XAF1 mRNA among all four cell lines.