RNA was converted to cDNA with Reverse Transcription System (Promega) according

to the manufacturer’s instructions. Q-PCR was performed using the miRNA SYBR Real-time PCR kit (Guangzhou RiboBio, Guangzhou, Guangdong, China) on the ABI 7300 Real-Time PCR system (Life Technologies, Grand Island, NY). To calculate relative expression, the (ΔΔCT) method was used in comparing miRNA expression in U251R cells to U251 parental cancer cells according to ABI’s protocol. Annexin V-FITC apoptosis detection This assay was performed according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, after treatment, cells were collected, washed selleck products with PBS and pelleted. Cell pellets were resuspended in 100 μL of Annexin V-FITC labeling solution and incubated at room temperature in dark for 30 minutes. After incubation, reaction was stopped by adding 300 μL ice-cold PBS and measured on FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Caspase-3 NVP-BEZ235 activity analysis Caspase-3 activity was measured by Caspase-Glo3/7 assay kit (Promega) according to the

manufacturer’s instructions. Cell cycle analysis This assay was performed as previously described [28]. Briefly, cells were harvested, washed twice with cold PBS and fixed with 70% this website cold ethanol overnight. Fixative was discarded and 0.2% Triton X-100 was added to the fixed cells. Cells were washed with PBS again and resuspended in PBS containing 50 mg/mL PI and 1 mg/mL RNase A for 30 min in the dark on ice. The samples were then analyzed on a flow cytometer. Statistics The Student′s t-test was used to compare the difference

between two tested groups. A value of p < 0.05 was considered as indicating a significant difference. Results Characterization of the induced cisplatin-resistant U251 cells 5-Fluoracil nmr We observed no apparent difference in morphology or growth rate between the parental U251 cells and cisplatin-resistant U251 cells (hereafter refers as U251R). To compare the sensitivity of the parental U251 and U251R cells to cisplatin, cells were treated with different concentrations of cisplatin for 72 hours and dose–response curves were plotted as shown in Figure 1A. Dose-dependent anti-proliferative activity were observed in both cell lines; however, the resistance of U251R to cisplatin was 3.1 fold higher than that of the parental U251 cells, as measured by the IC50 values for cisplatin over 48 hours treatment: 1.4±0.1 μg/mL and 4.4±0.9 μg/mL, respectively (Figure 1B). Figure 1 Characterization of the induced cisplatin-resistant U251 cells. (A) U251 and U251R cells were treated with indicated concentration of cisplatin for 72 hours and cell viability was tested by MTT. (B) IC50 of cisplatin in U251 and U251R cells was calculated.