Fig. 4.

Effects of siRNA-mediated down-regulation of treacle on rRNA production. (A) HeLa cells were treated with 40 nM siRNA or mock-transfected. si1011 targets the TCOF1 mRNA and si934Scr is a control siRNA. After 48 h of transfection, total RNA was isolated and analyzed by RT-PCR using TCOF1-specific primers and U1C primers as an internal control. In a separate 4-day transfection, cells were boiled in Laemmli buffer and analyzed by Western blot enhanced chemiluminescence using anti-treacle antibody. The blot was stripped and reprobed with anti-Guα antibody. Treacle comigrates at ≈220 kDa near the top of the gel, whereas RNA helicase II/Guα comigrates near the bottom of the gel at ≈90 kDa. (B) An RNase protection assay shows a 47% decrease in the level of pre-rRNA in cells treated with si1011 compared to mock-transfected cells. Lane 1 contains 1/10 of the riboprobe used in the other lanes. Lanes 2–5 contain 8 μg yeast RNA or total RNA isolated from cells mock-treated or treated with the indicated siRNA. (C) Total RNAs (1.0 μg) from ³²P-metabolically labeled transfected cells were analyzed by gel electrophoresis. The chase times with nonradioactive phosphate are labeled 0, 0.5, and 1 h. The numbers below correspond to the amount of specific RNA band relative to mock-transfected samples (set at 100) obtained with image-quant software. (D) The same blot was stained with 0.2% methylene blue to visualize total RNA. (E) BrUTP incorporation assay. HeLa cells grown on a chamber slide were transfected twice with 40 nM si1011 (24 h apart) and analyzed for BrUTP incorporation 72 h after the last transfection. Mock- and si1011-transfected cells were indirectly stained with anti-treacle (green) and anti-BrdUrd (red) antibodies. All pictures were taken at a preset brightness, contrast, and gamma level of 50, 50, and 1.0, respectively, and image scale range of 100–3,000. (F) Fluorescence intensity profiles of the images shown in E.