Fig. 1. Human HCF-1 is depleted efficiently and specifically by siRNA. (A) Schematic structure of HCF-1 (Wysocka et al., 2001a). Above are shown (i) structural or sequence elements and (ii) functional regions. The position of the tsBN67 P134S missense mutation is indicated. The selected HCF-1 coding region RNA sequence (nucleotides 124–144; Wilson et al., 1993) and the corresponding HCF-1 siRNA duplex are indicated below. (B) HCF-1 (top) and β-actin (bottom) mRNA levels in untreated HeLa cells (lane 1) or 3 days after treatment with either control (lane 2) or HCF-1 (lane 3) siRNA. (C) Immunoblot analysis of HCF-1N (top), HCF-1C (middle) and β-actin (bottom) proteins of untreated HeLa cells (lanes 1) or 3 days after treatment with control (lanes 2) or HCF-1 (lanes 3) siRNA. (D) Control (a and b) or HCF-1 (c and d) siRNA-treated cells were subjected to analysis after paraformaldehyde fixation by nuclear DAPI (a and c) and αHCF-1N (b and d) staining. Arrowheads point to cells exhibiting HCF-1 depletion. Scale bar: 5 µm. (E) Percentage of HCF-1-positive cells after one or two transfections with control or HCF-1 siRNA as determined by immunofluorescence. The percentage of HCF-1-positive nuclei was obtained by counting 200 cells per sample in each of three independent experiments.