e149 Nucleic Acids Research, 2007, Vol. 35, No. 22 PAGE 4 OF 9 Figure 1. Continued. of K562/S-miR17/19b-IEW, K562/MSCV-c-myc- IRES-EGFP-WPRE, K562/pGL3-miR-18a-sensor, K562/pGL3-miR-18a-control, K562/pGL3-miR-19b- sensor, K562/pGL3-miR-19b-control and K562/pGL3- miR-20a-sensor, K562/pGL3-miR-20a-control cells each of the latter (K562/pGL3), expressing luciferase as reporter gene as described (7). Lentiviral transduction was per- formed twice as recently described with an MOI of approximately two (14). For combined expression of miRNAs and antagomirs, K562 cells were ﬁrst transduced with lentivirus-encoded antagomirs. After 72 h (=day 0), miRNAs were over- expressed, and cell counting was performed at the time points indicated in Figure 2E. Anti-c-myc-shRNAs corresponding to position 817–835 of the human c-myc gene (Gene bank accession no. NM_002467) (7), lentiviral transgene plasmids pdc- SR and shRNA controls were cloned as described (14). RNA isolation, miR-qRT-PCR and northern blot Total RNA from K562 cells was prepared using Trizol (Invitrogen). Expression of mature miRNAs was deter- mined by miR-qRT-PCR using miRNA-speciﬁc looped RT-primers and TaqMan probes as recommended by the manufacturer (Applied Biosystems, Foster City, USA). Normalization was performed using the 2- CT method relative to miR-16 miRNA, which was nearly equally expressed in K562 cells in all conditions tested (7, and data not shown). PCR was performed in duplicate using an ABI7500 cycler. To study the impact of oligonucleotide annealing on miR-qRT-PCR in vitro, we performed an annealing reaction by heating total RNA (10 ng) in the presence of miRNA-complementary oligonucleotides (0.2 mM/l) for 10 min at 708C followed by cooling down to 168C before conventional RT was performed according to the manu- facturer. In some experiments shown in Figure 1E, a second heating step for 10 min at 708C was performed for re-denaturation. For northern-blotting, total RNA (10 mg) was separated by electrophoresis using an 16% denaturing polyacryla- mide gel and subsequently transferred by electroblotting onto Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Hybridization and wash- ing were carried out at 378C according to standard protocols. Membranes were probed with 32P-labeled 22– 23-nt oligonucleotides corresponding to the respective miRNA, miR-18a and miR-20a and were subsequently visualized by autoradiography. Endogenous U6 snRNA served both as an internal size standard and a loading control (U6sn probe: 50-TATGGAACGCTTCACGAA TTTGC-30). Luciferase assay Luciferase assays were performed 96 h after transduction using the Luciferase Reporter Assay System (Promega). Fireﬂy luciferase activity was normalized to cell numbers for each reaction. Cell proliferation of transduced K562 cells A total of 5 104 transduced K562 cells/ml were cultured in 24-well plates and viable cells were counted from 1 to 14 days by trypan blue exclusion. Immunoblotting Cellular lysates from K562 cells were prepared with lysis buﬀer (250 mM Tris–HCl, pH7.5, 0.5% Triton X-100; 10 mM NaF; 10 mM Na3VO4; 1 mM PMSF; 2 mM EDTA; 2 mM AEBSF; 2 mM Aprotinin; 10 mM Bestatin; 3 mM E- 64; 4 mM Leupeptin; 2 mM PepstatinA) separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to Hybond-enhanced chemilu- minescence (ECL) nitrocellulose membrane (Amersham Bioscience, Uppsala, Sweden), and incubated with mono- clonal mouse anti-E2F-1 (KH20 + KH95, upstate, biomol, Hamburg) and monoclonal mouse anti-a-tubulin antibo- dies (DM1A, Calbiochem, Schwalbach am Ts.) according to the manufacturer’s protocol. Chemiluminescence was