Figure 3

Dicer knockdowns do not activate endogenous CR1 transcripts in Dicer-deficient DT40 cells. (A, C) Strand-specific RT-PCR targeting the sense message of CR1 elements was performed to determine the relative levels of CR1-B and CR1-F elements by primer sets that detect CR1's ORF1 and ORF2 from Dicer⁺ (Dox-), and Dicer-deficient (Dox+) cells. Digital photographs (negative image) of agarose gel analyses of the RT-PCR products are shown including the transcripts of CR1-B, CR1-F, Dicer, and chicken β-actin (internal control). (B, D) Quantitative real-time RT-PCR analysis of CR1-B and CR1-F transcripts were determined after normalizing the data with chicken β-actin. Error bars show s.d. (n = 6).(E) A schematic of the constructs used for the luciferase reporter assay is shown in the top panel. Luciferase reporter activity of CR1 5'UTR compared with L1 5'UTR after normalized for Renilla luciferase reporter is shown in bottom panel. In this assay, the negative control GL3 vector was used. Error bars show s.d. *P < 0.001. (F) Relative luciferase activity of CR1 and L1's 5'UTR in chicken DT40 cells measured in the presence or absence of Dicer. All activities were normalized to that of Renilla luciferase reporter and arbitrary units of each relative luciferase activity converted into the percentage. *P < 0.005 and **P < 0.001.