Figure 4

A. Total RNA isolated from HEK293T cells transfected with either a "classic" or "YIU" construct targeting luciferase was analyzed by Northern blot hybridization to a radioactively labeled luciferase 19-nt sense-strand oligonucleotide and to a 19-nt oligonucleotide complementary to 5S RNA. 19- and 27-nt single-stranded DNA oligonucleotides were used as size standards; because DNA rather than RNA oligonucleotides were used, the indicated sizes are only approximate. The expected approximate sizes of the hairpin transcripts are 49 nt ("classic") and 62 nt ("YIU"), whereas both processed forms are expected to be ~21 nt. B. and C. Constructs expressing luciferase mRNA fused with a fragment of CCND1 mRNA were cotransfected with shRNA expression constructs targeting either luciferase or CCND1, as described in Table 1. The negative control shRNA expression construct was a "classic" construct targeting BCL2. Samples were analyzed in duplicate, and values were normalized using Renilla luciferase expression. Error bars represent the standard deviation. The shRNA constructs targeting CCND1 was cotransfected with a luciferase-CCND1 fusion construct containing the target sequence, with the exception of #3, whose target sequence is absent from both fusion constructs. Construct #3 thus serves as an additional negative control. The small degree of suppression seen with #3 and the apparent small stimulation by some shRNA cassettes may be either due to experimental error or to off-target effects of #3 and of the control shRNA construct. The data shown are representative of three individual experiments, which all showed similar results.