The cultured cells were randomly divided into three groups: control group (0 Gy, without the embedded seed in the paraffin), 2 Gy, and 4 Gy. Apoptosis analysis by flow cytometry Adherent SW 1990 cells cells were trypsinized

and centrifuged for 5 min at 220xg. Cells were then washed three times in ice cold PBS and suspended in binding selleckchem buffer (0.01 M Hepes, pH 7.4; 0.14 M NaCl; 2.5 mM CaCl2) at 1 × 106 cells/ml. The cells were stained with annexin V-FITC (1 μl/ml) and propidium iodide (5 μg/ml) for 15 min in the dark as described previously [15]. Cells were analyzed by fluorescence-activated cell sorting (FACS) using a Coulter EPICS and MOdFit SOFTWARE (Verity Software House, Topsham, MN). Each test was performed 3 times. Real-time polymerase chain reaction (PCR) Total RNA was retracted from SW 1990 cells using Trizol

reagent (Invitrogen, Carlsbad, CA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase GAPDH was used as an internal reference [16]. Real-time PCR was performed by using the following primers: learn more for DNMT1, upstream primer 5′-GTGGGGGACTGTGTCTCTGT-3′ downstream primer 5′-TGAAAGCTGCATGTCCTCAC-3′, and amplified NVP-BSK805 mouse fragment length of 204 bp; for DNMT3a, upstream primer 5′-ATCTCGCGATTTCTCGACTC- 3′, downstream primer 5′-GCTGAACTTGGCTATCCTGC -3′, and amplified fragment length of 180 bp; for DNMT3b, upstream primer 5′-TTGAATATGAAGCCCCCAAG- 3′, downstream primer 5′-TGATATTCCCCTCGTGCTTC -3′, amplified fragment length of 160 bp; for GAPDH, upstream primer 5′-GCACCGTCAAGGCTGAGAAC-3′, downstream primer 5′-ATGGTGGTGAAGACGCCAGT-3′, amplified fragment length of 142 bp. Cycling parameters: pre-denaturation 1 min, 95°C; denaturation 15 s, 95°C;

annealing 15 s, 60°C; extension 45 s, 72°C, 40 cycles; final extension 5 min, 70°C. The PCR was repeated three times for each sample. The standard curve was generated with the ABI 7500 Real Time PCR system (Applied Biosystems, Carlsbad, CA, USA) to describe the linear relationship between threshold cycle (Ct) value and relative quantity (RQ). RQ values were obtained from measured Ct value with the following formula: 2(-ΔΔCt), where ΔΔCt = ΔCtT; ΔCtS = (ΔCtT – ΔCtTE ) – (ΔCtS – ΔCtSE), T is the target sample, S is the SW-1990 cell sample, and E is the reference. The RQ of mRNA in all groups were calculated relative to the RQ value in control group 1. Western blotting Western blotting Isoconazole was performed as described previously [17, 18]. Nuclear protein was prepared from SW-1990 pancreatic cancer cells with a Nuclear Protein extraction kit (Fermentas, Ontario, CA). The total protein concentration was determined by the Bradford assay using the Coomassie Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL). Prepared protein samples (20 μg each) were boiled for 5 min and loaded onto a 12% SDS polyacrylamide gel. After separation by electrophoresis and electroblotting to nitrocellulose membranes, membranes were blocked by with 5% nonfat dry milk in 0.