Supplemental methods
DNA Constructs and reagents
The NF-кB p65 and p50 expression plasmids were used to produce full-length p65 and p50 protein. It was made by cloning PCR products into the HindIII and EcoRV sites of pFlag-CMV-2expression vector as described before. (Hertlein E et al. 2005).
The NF-кBactivity reporter which contains three tandem repeats of the wild-type κB site, from the major histocompatibility complex (MHC) class I enhancer fused to the luciferase reporter gene were along (Guttridge, et al., 1999). The NF-кB(p50) and (p65) plasmids, the potent and NF-кBspecific inhibitor IκBα-SR(Brockman JA et al, 1995) and the NF-кBreporter were kindly provided by Dr. Dennis Guttridge ( the OhioStateUniversity).
MiRNA quantitative stem-loop RT-PCR. We usedsingle tube TaqMan miRNA assays as previously described (Chen et al., 2005) (Applied Biosystems, Foster City, CA). Normalization was performed with 18S. All RT reactions, including no-template controls and RT minus controls, were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Gene expression levels were quantified using the ABI Prism 7900HT Sequence detection system (Applied Biosystems). Comparative real-time PCR was performed in triplicate, including no-template controls, using specific primers for mir-15a, miR-16-1, mir-223, miR-107, miR-181b, let-7a, let-7c, let-7b and miR-342 (all reagents obtained from Applied Biosystems). Relative expression was calculated using the comparative Ct method.
Taqman Real Time PCR. Comparative real- time RT-PCR was performed for Bcl-2 and Ras in triplicate. Primers and probes were obtained from applied Biosystems (Foster City, CA). Gene expression levels were quantified using ABI Prism 7900 Sequence detection system (Applied Biosystems). Relative and fold difference expression were calculated using the Ct and 2(-delta Ct) method after normalization with 18S.
Celltransfection. NB4 cells were seeded at 5 X 106 and transfected using nucleoporation (AMAXA, Kit V, and program T-019) with 10 ug of miR-16-1 orlet-7a-3. NB4 cells transfected with scrambled oligonucleotides(negative control 1, AMBION) were used as controls.