Data analysis was per formed using SDS 2. 3 software, which utilizes the delta delta CT method. Real time quantitative reverse transcription PCR Total RNA was prepared from OS tissues or cell lines using TRIzol reagent followed by purification with TURBO DNA free System. The cDNAs were synthesized using SuperScript II reverse transcriptase. Real time quantitative PCR was performed using SYBR Green PCR master mix in a 7300 Real time PCR System. TaqMan microRNA assays that include RT primers and TaqMan probes were used to quantify the expression of mature miRNA 33a. The mean Ct was determined from triplicate PCRs. Gene expression was calculated relative to GAPDH. For measurement of TWIST mRNA, the following primers were used, for human TWIST, and. The results were normalized against that of the GAPDH gene in the same sample.

Each experiment was repeated for two times in triplicates. Western blot analysis Briefly, cells were dissolved in 250 ul of 2× SDS loading buffer, and in cubated at 95 C for 10 min. Equal amount of proteins CX-6258 cancer for each sample were separated by 10% SDS polyacrylamide gel and blotted onto a polyvinylidene difluoride micropor ous membrane. Mem branes were incubated for 1 h with a 1 1000 dilution of primary antibody, and then washed and revealed using secondary antibodies with horseradish peroxidase conjugate. Peroxidase was revealed with a GE Healthcare ECL kit. Transfection and lentiviral transduction Plasmid constructs were transfected into cells using Superfect transfection reagent according to the manufactures instructions.

Pools of stable transfec tants of TWIST were generated via selection with G418 by the manufacturers protocol. Lentiviral transduction of TWIST shRNA was performed and pools of stable transductants were generated via selec tion with puromycin. Luciferase assay MG 63 cells were transfected with luciferase reporter {in the know|Micafungin Sodium ntifungal drug con structs using Superfect transfection reagent. Lu ciferase activity was measured 72 hours after transfection using the Dual luciferase reporter assay system following the manufacturers instructions. Experiments were conducted in triplicates and results were expressed as ratios between renilla and firefly luciferase counts. Measurement of apoptosis by TUNEL assay The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System by the manufacturers protocol. Cells were treated with cisplatin for 8 hours. Apoptotic cells exhibit a strong nu clear green fluorescence that could be detected using a standard fluorescein filter. All cells stained with DAPI exhibit a strong blue nuclear fluorescence. The slides were observed under fluorescence microscopy with relative apoptotic cells determined by counting TUNEL positive cells in five random fields for each sample.