SiRNA and shRNA knock-down targeted to endogenous pre-mRNAs.

<p>(A) & (B) The targeted regions on the RNAs for each of the snoMEN vectors used in this study are shown in a schematic diagram. The same pre-mRNA sequence of UBF1 (A)/SMN1 (B) as targeted by the snoMEN vector was targeted by siRNA oligoribonucleotides and shRNA expression plasmids. (C) Western blot analysis for siRNA experiments. Detection of endogenous UBF1 protein levels following transfection of HeLa cells using either Scrambled siRNA (Control: lane1), UBF1 siRNA (siUBF1: lane2), UBF M box siRNA-1 (siUM1: lane3), UBF M box siRNA-2 (siUM2: lane4), and UBF M box siRNA-3 (siUM3: lane5). An equivalent amount of HeLa extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-UBF1 antibody and with an anti-tubulin antibody as a loading control. Graph shows UBF1 signal intensity normalised to the tubulin signal measured from three independent experiments. (D) Structure of shRNA expression plasmids. A cDNA producing a short hairpin RNA was subcloned under the U6 RNA polymerase III promoter. Each of the shRNA plasmids encode ZsGreen FP–protein cDNA under CMV promoter regulation as a transfection marker. (E) UBF1 shRNA plasmid and no-endogenous target shRNA plasmid were transfected as a positive and negative control, respectively. UBF1 Mbox shRNA-1 to -3 (shUM1–3) have the same target sequence as UBF1 snoMEN from set1 to set3, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062305#pone-0062305-g003" target="_blank">Figure 3A</a>). Scale bar, 10 µm. Arrow: cells not showing knock-down, Arrowhead: cells showing knock-down. (F) Western blot analysis for shRNA experiments. Detection of endogenous UBF1 protein levels following transfection of HeLa cells using either Scrambled shRNA (Control: lane1), UBF1 shRNA (shUBF1: lane2), UBF M box shRNA-1 (shUM1: lane3), UBF M box shRNA-2 (shUM2: lane4), and UBF M box shRNA-3 (shUM3: lane5). An equivalent amount of HeLa extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-UBF1 antibody and with an anti-tubulin antibody as a loading control. Graph shows UBF1 signal intensity normalised to the tubulin signal measured from three independent experiments.