Louis, MO). Cell survival assays Briefly, cells were seeded at an initial density of 5 × 104 cells/ml in a 96-well plate for 24 h. After transfection, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added into each well at a final concentration of 0.5 mg/ml. The insoluble formazan was collected,
dissolved in dimethylsulfoxide and measured with an ELISA reader (Bio-Rad, USA) at a wavelength of 570 selleck nm. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) RNA isolation was performed using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. SuperScript Preamplification System (Gibco BRL, Gaithersburg, MD) was used for cDNA synthesis. Two microgramme of cDNA was used as a template for PCR reaction. The following primers were used: GAPDH: forward 5′-GTC AGT GGT GGA CCT GAC CT-3′ and reverse 5′-AGG GGT CTA CAT
GGC AAC TG-3′; p53: forward 5′-TAC TCC CCT GCC CTC AAC AAG A -3′ and reverse 5′-CTT AGC ACC TGA AGG GTG AAA TAT TC-3′, and NDRG2: forward 5′- ATG GCG GAG CTG CAG GAG GTG-3′ and reverse 5′-AAC AAG GGC CAT TCA ACA GGA GAC-3′. The cycling conditions were as follows: initial denaturantion (5 Ro 61-8048 order minutes at 94°C), followed by the appropriate number of 26 cycles of denaturation (94°C, 30 seconds), annealing (GAPDH, 30 seconds at 60°C; p53, 30 seconds at 65°C; NDRG2, 30 seconds at 68°C) and elongation (30 seconds at 72°C), and a final extension (10 minutes at 72°C). The samples were visualized by electrophoresis in 1.2% agarose gel and ethidium bromide. Cell Cycle and apoptosis Analysis Flow cytometry see more analysis was performed as described. Cells were seeded overnight PRKD3 on 60-mm-diameter plates in a complete medium, placed in a serum-free medium for 48 hours to synchronize the cells, and then kept again in the complete medium. At 24 hours, cells were recovered. After washing with ice-cold PBS, cells were suspended in about 0.5 ml of 70% alcohol and kept at 4°C for 30 minutes.
The suspension was filtered through a 50-mm nylon mesh, and the DNA content of stained nuclei was analyzed by a flow cytometer (EPICS XL; Coulter, Miami FL). Cell cycle was analyzed using Multicycle-DNA Cell Cycle Analyzed Software (FACScan, Becton Dickinson, San Jose, CA). The proliferous index (PI) was calculated as: PI = (S + G2)/(S + G2 + G1). Apoptosis index was measured using Annexin V-FITC apoptosis detection kit (Sigma) and subsequently analyzed by flow cytometry. Each experiment was performed in triplicate [13, 14]. Statistical Analysis All statistical analyses were performed using the SPSS 16.0 statistical software package (SPSS, Chicago, IL). The differences in apoptosis index between groups were compared using one-way analysis of variance, and data were expressed as mean ± SEM. Statistical difference was accepted at P < 0.05.