Figure 6.

Effect of chemotherapeutic drugs on HeLa cells treated with E2-EPF siRNA. Cells were transfected with the indicated negative control siRNA (NTp, MYOGp, NT) and E2-EPF siRNA (E2-EPFp, D5, D6) and treated in duplicate (A) or in triplicate (B and C) with the indicated drug concentrations (A and C) or with no drug (B) for 3 days starting 24 hours after transfection. Cell proliferation (nuclei counts) was measured by microscopic imaging analysis. (B) Left: Nuclei counts for “no drug” control transfectants corresponding to the experiment in (C). Right: Samples (40 µg protein/lane, except as noted) on the left were analyzed by immunoblotting for the E2-EPF protein at 24 and 72 hours after transfection. Data are representative of at least three independent experiments. Error bars, S.D. *P < .03 for the comparison of E2-EPF siRNA with negative control siRNA. Note: D6 + dox was not statistically significant (P > .05), but in the repeat experiment, NT + dox versus D6 + dox yielded 63% vs 37% (5 nM) and 28% vs 14% (10 nM) (P = .014). (D) HeLa cells were transfected with the indicated siRNA, harvested 48 hours later, and fractionated into nuclear and cytoplasmic extracts before immunoblot analysis (50 µg protein/lane) for topo IIα (170 kDa) and topo IIβ (180 kDa) expressions. Immunoreactive bands identified in topo IIα and IIβ siRNA-treated cell lysates indicate the specificity of the topo II antibody. Quantification of topo IIα by protein dilution in an independent experiment demonstrated that the increase in topo IIα on E2-EPF knockdown was ∼ 2-fold. Amido black staining verified equivalent sample loading.