Figure 3.

BLM deficiency slows RNA polymerase I-mediated 45S rRNA transcription rate. (A) 293T cells transfected with either an αBLM-directed siRNA or scrambled control siRNA were pulse-labeled with ³H-uridine for 30 min (P) and chased for 1 h (C) with cold uridine. Isolated RNAs were separated on a 1% MOPS-formaldehyde agarose gel, transferred to a nylon membrane and analyzed by autoradiography. 45S, 28S and 18S rRNA species are indicated next to autoradiograph (top panel). Ethidium bromide staining demonstrates equal loading of RNA (middle panel). Western blot demonstrates efficiency of BLM knockdown; lamin B serves as a protein loading control (bottom panel). (B) ³H-uridine pulse-chase analysis in BS fibroblasts (GM08505) transfected with either pGFP–BLM or pGFP; figure is labeled as in (A). (C) The pulse-chase analyses in 293T and GM08505 cells were analyzed using ImageQuant software to measure the 45S rRNA transcript abundance in the BLM-proficient cells (293T scrambled siRNA-transfected or GM08505 pGFP–BLM-transfected) compared with the BLM-deficient cells (293T αBLM-directed siRNA-transfected or GM08505 pGFP-transfected).