FIG. 4.

Distinct changes in ploidy are linked with KIF14 siRNA efficacy. (A) HeLa cells transfected with KIF14-specific moderate and strong siRNAs, M-1, M2, S-1, S-2, and S-3, were analyzed by flow cytometry 72 h after transfection. The DNA content of KIF14-specific siRNA-transfected cells is compared to that of cells transfected with a luciferase-specific siRNA. (B) Cells transfected with either of two polyploid-inducing KIF14-specific siRNAs (S-1 or S-2) were analyzed by immunofluorescence microscopy at 72 h after transfection. Staining with α-tubulin and Hoechst allowed the evaluation of nuclear morphology of single cells. A total of 100 cells were evaluated for each siRNA duplex, and the number of mononuclear, multinuclear, and mitotic cells was compared to those of a luciferase (Luc) siRNA control. (C) HeLa cells were transfected with a panel of KIF14-specific siRNAs linked to different ploidy phenotypes as described in Fig. 4A. Cells were harvested 24 h after transfection, and equal amounts of protein were analyzed by immunoblotting with anti-KIF14 antibody. Cells transfected with an siRNA targeting luciferase, a gene not expressed in mammalian cells, served as the negative control. Those siRNAs that reduced KIF14 expression by less than 50% are labeled as moderate. Those siRNAs that reduced KIF14 expression more than 80% are labeled as strong. The percent of KIF14 remaining for each sample was calculated using Oct1 as a load control.