The isopropanol was added to the cells, absorbance was measured in an ELISA reader at 570 nM

copies of complementary sequences against miRNA-16 were inserted after the stop codon of the hNIS/Fluc fusion gene to generate GV260-hNIS/Fluc3xmir16. A scrambled nucleotide sequence of similar length to 3xmir16 was also inserted at the 39UTR of hNIS/Fluc fusion gene to obtain a control construct were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 100 units penicillin and 100 mg/mL streptomycin. To maintain the MDR phenotype, vincristine was added to the culture medium for SGC7901/ VCR cells. To generate stable cell line, lentivirus vector GV260hNIS/Fluc or GV260-hNIS/Fluc-3xmir16 was first cotransfected into 293T cells with packaging plasmids. Growth medium was changed at 6 h post-transfection and lentiviruscontaining supernatant was harvested at 48 h post-transfection. Harvested supernatants were centrifuged at 4,000 g for 10 min to pellet cell debris. Concentrated virus was titrated on 293T cells. SGC7901 cells and SGC7901/VCR cells were transduced with GV260-hNIS/Fluc and GV260-hNIS/Fluc-3xmir16 virus respectively at a multiplicity of infection of 10. Then the cells were screened with puromycin for 3 weeks and cell colonies were collected to 6145492 expanded for next step G5555 experiments. Reagents and antibodies Mouse monoclonal antibody against hNIS was purchased from Abcam. Rabbit polyclonal antibody specific to Bcl-2 was purchased from Santa Cruz Biotechnology. Bay11-7082 and SB203580 were obtained from Beyotime Institute of Biotechnology. The anticancer drugs including vincristine, etoposide, mitomycin C, cisplatin, 5-fluorouracil and Adriamycin were purchased from Wolsen Biotechnology. The GV260-Fluc-puro lentivirus plasmid was obtained from Shanghai GeneChem Company. All cell culture media and serum were purchased from Gibco. For the construction of a dual expression vector, the cDNA of hNIS gene was inserted into the BamH I and Age I restriction enzyme sites of a lentiviral vector GV260-Fluc-puro that encodes a Fluc gene under the control of the ubiquitin promoter. The resulting dual expression construct Quantitative real time PCR analysis Total RNA was isolated from the cultured cells using Trizol. RT was performed with PrimerScript RT Regent Kit and qPCR was performed with Fast SYBR Green Master Mix and ABI Noninvasive Visualization of MicroRNA-16 StepOne Plus Real-time PCR system according to the manufacturer’s protocol. PCR products were analyzed on 3% agarose gels. The relative amount of miRNA-16 was normalized to U6 snRNA. And the relative amount of Fluc was normalized to GAPDH gene. The fold-change for miRNA-16 or Fluc gene from experiment group relative to the control group was calculated using the 22DDCt method, where DDCt = DCt experiment – DCt control and DCt = Ct miRNA – Ct U6 or DCt = Ct Fluc – Ct GAPDH. Cell lysates were then centrifuged at 12,000 rpm for 15 min 7751958 at 4uC. The supernatant was collected and identical amounts of protein were resolved by 8% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated in blocking solution containing 5% BSA for 1 h at room temperature, and then immunoblotted with indicated antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence kit. MTT assay To determine the growth rates of NF-empty, NF-3xmir16 and NF-3xmir16/VCR cells, these three types of cells were inoculated into 96-well plates with the same numbers per well. At different time points, 25 ml of 5.0 mg/ml sterile filtered 3–2, 5- diphenyl tetrazo