R adenovirus amplification. K562 cells, a cell line established from a chronic myelogenous leukemia patient in blast crisis, were cul-Page 2 of(page number not for citation purposes)Journal of Experimental Clinical Cancer Research 2008, 27:http://www.jeccr.com/content/27/1/for 30 minutes before the addition of propidium iodide (PI), and then placed at 4 for more than 15 minutes. Finally, the cell cycle distribution (G0/G1 phase, S, G2/M) and cell apoptosis rate was detected by FCMMeasurement of the target genes by FQ-PCR K562 cells were collected from each group, washed with PBS three times. Total RNA was extracted from the cells according to the RNeasy Mini Protocol (Qiangen Biothch, Beijing). Each 1 g total RNA from each group of K562 cells was reversely transcribed in a 20 L reaction mixture which contained 0.5 g oligo (dT)15 as primer by SuperScript II reverse transcriptase (Promage). The resultant cDNA was subjected to FQPCR. PCR was carried out in a total 25 l reaction system, with the SYBR Green I PCR Reagent kit (Qiangen Biothech, Beijing) and a Lightcycler fluoresencent quantitative amplification analyzer (BioRad, Hercules, CA). The reaction condition was 94 for 5 min, 94 45s and 60 1 min, for 30 cycles. The BX795 supplement fluorescence signal of the first 3 to 15 cycles was settled as fluorescent background signal and the baseline was adjusted accordingly. Relative quantification of gene expression level was calculated by the following method: for each reaction to amplify the target gene and -actin gene, the Ct value was determined. The PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 ratio of of each target mRNA to -actin mRNA was calculated by the formula 2-Ct. The specific primers’ sequences for the target genes were shown in Table 1. Measurement of the target proteins by western blotting Western blot analysis was performed for detecting the expression level of specific protein in freshly washed K562 cells from each group. The protein concentrations of the cell lysate were measured by coomassie brilliant blue kit (Kang Chen Biotechnology, Beijing, China). Each 50 g of the cell lysate was loaded onto a 10 SDS-PAGE gel, and run at 100 volts for 2 h. Cell proteins were transferred to nitrocellulose membrane, blocked with 5 milk in TBS (Tris-buffered saline with 0.1 Tween 20) for 1 h, then,Table 1: Primer sequence for FQPCRwashed and incubated with the primary antibody for 1 h at room temperature or overnight at 4 . The blots were washed and incubated with the horseradish peroxidaseconjugated secondary antibody, and developed with a chemiluminescent substrate, ECL Plus. Following development of the primary antibodies, the bounded immunoglobulins were removed from the membranes by washing twice, each time for 15 minutes at room temperature in Restore Western blot Stripping Buffer. An autoradiograph was obtained, and protein levels were measured using a Fluors scanner and Quantity One software for analysis (Bio-Rad).Statistical analysis tatistical analysis was done by Student’s t-test and Spearman’s test. Data was shown by mean ?SD of three independent experiments. The results from each group were compared with that of the corresponding control. P < 0.05 was considered as statistically significance.ResultsAnti-proliferation effect of PTEN transfection in K562 cells Our preliminary experiment and several published papers showed that K562 cells expressed PTEN mRNA and protein at very low levels [8]. Therefore, K562 cells were transfected with Ad-PTEN-GFP or Ad-GFP in the present study.
