RNA interference with HRPT2 or Paf1 expression in human cells leads to down-regulation of endogenous Paf1 and Leo1 expression and increased cell proliferation. (A and B) Immunoblotting analysis of parafibromin (Top Panel), Paf1 (Second Panel), Leo1 (Third Panel) in HeLa cells following RNAi-mediated depletion of HRPT2 or Paf1 transcripts. Cells were treated with control siRNA or one of two HRPT2- or Paf1-specific siRNAs and analyzed as described in Methods. The expression of β-actin is shown as loading control (Fourth Panel). (C and D) Inhibition of parafibromin or Paf1 expression by RNAi resulted in increased S phase cell population. HeLa cells were treated with control siRNA or one of two HRPT2- or Paf1-specific siRNA and subjected to FACS analysis as described in Methods. (D) The histogram shows the distribution of different cell cycle population; values are representative of two independent experiments with similar results. Significance of the differences on S phase and G1 phase were evaluated by Student's t test (***, P < 0.001). (E and F) Proliferation analysis. Cultured HeLa (E) or NHDF cells (F) were treated as above for 48 h. Cell proliferation was estimated by a colorimetric cell proliferation assay. Presented are data representative of three individual repeats. Significance of the differences were evaluated by Student's t test (*** P < 0.005, **, P < 0.01, *, P < 0.05).