Skip to main content
. 2013 Dec 19;9(12):e1003818. doi: 10.1371/journal.ppat.1003818

Figure 3. TTV tth8 miRNAs are generated by the canonical host miRNA biogenesis pathway.

Figure 3

(A) Northern blot analysis of RNA from HEK293 cells transfected with MHV68-miR-M1-7 expression vector, SV40-miR-S1 TTV recombinant genome preparation, or wildtype TTV-tth8 genome preparation with or without treatment with the RNA pol II inhibitor α-amanitin. Ethidium bromide stained low molecular weight RNA is shown as a load control. (B) Northern blot analysis of RNA from HEK293T cells co-transfected with MHV68-miR-M1-7, SV40-miR-S1, or TTV-tth8-miR-T1 expression vectors and either Drosha siRNA (siDrosha) or negative control siRNA (siNC). Ethidium bromide stained low molecular weight RNA is shown as a load control. (C) Northern blot analysis of RNA from either DLD1 (WT) or DLD1DicerEx5- (DicerEx5-) cells transfected with either MHV68-miR-M1-7 or TTV-tth8-miR-T1 expression vectors. Ethidium bromide stained low molecular weight RNA is shown as a load control. (D) RISC reporter assay for TTV-tth8-miR-T1. HEK293 cells were co-transfected with either empty miRNA expression vector (Vector) or TTV-tth8-miR-T1 expression vector (TTH8) and both control firefly luciferase reporter and Renilla luciferase based reporter plasmids with vector UTR (Vector), two sites perfectly complementary to TTV-tth8-miR-T1-5p (5p), two sites perfectly complementary to TTV-tth8-miR-T1-3p (3p), or control 3′UTR reporter with GAPDH 3′UTR (GAPDH). The average relative Renilla luciferase activity normalized to firefly luciferase activity of three replicates is shown. Error bars indicate SD of three replicates and p-values were calculated using Student's t-test.