PAGE 3 OF 9 Nucleic Acids Research, 2007, Vol. 35, No. 22 e149 B C Figure 1. Lentivirus-mediated antagomir expression. (A) Schematic representation of the lentiviral transgene plasmid harboring an antagomir expression cassette with a human H1-RNA promoter inserted into the U3 region of the 30LTR. This location results in duplication during reverse transcription indicated as ‘double-copy’ (dc) vector. S:SFFV-LTR, E:EGFP, W:WPRE (B) Inhibition of miR-18a, miR-19b and miR-20a function by speciﬁc antagomirs in luciferase reporter assays. Control- (dark gray bars) or anti-c-myc- (light gray bars) shRNA-expressing K562 cells were stably transfected with miR-18a sensor (upper panel), miR-19b sensor (middle panel) and miR-20a sensor (lower panel) luciferase constructs, respectively. Reporter K562 cells were lentivirally transduced with ant-ctrl, anti-miR-18a, anti-miR-19b and anti-miR-20a antagomirs, respectively, as indicated. The ratio of normalized sensor to control luciferase activity is shown as described (7,16). Mean of two independent experiments are shown. (C) miRNA-speciﬁc quantitative RT-PCR (miR-qRT-PCR) for miR-18a, miR-19b and miR-20a, and the polycistronic pri-miR 17-92 transcript in the presence of lentivirally expressed antagomirs anti-miR-18a, anti-miR-19b, anti-miR-20a as compared to control (ctrl)-antagomir. Mean of two independent experiments are shown. miR-16 served as an endogenous control. (D) Northern blot analysis of miRNA expression in the presence or absence of lentivirally expressed antagomirs (miR-18a left panel, miR-20a right panel). U6 snRNA served as loading control. (E) miR-qRT-PCR of miRNAs in the presence of oligonucleotides complementary to the entire miRNA sequence (A), the 50- (B) and 30-half (C), respectively. The table indicates fold changes of miRNA levels as determined by miR-qRT-PCR. (F–H) Cell proliferation of wild-type K562 cells (F), K562 cells over-expressing the miR-17-19b cluster (G), or c-MYC (H), upon lentiviral transduction with anti-miR-18a, anti-miR-19b and anti-miR-20a antagomirs as compared to anti-ctrl transduced cells. K562 cells were lentivirally transduced (transduction eﬃcacy >95%, data not shown), plated at 104 cells per well two days after transduction, and the number of trypan blue negative cells was counted. Mean of two independent experiments are shown. The highest cell number on day 14 was set 100%.