Eg5 knockdown inhibits PAUF secretion in HeLa cells. HeLa cells were transfected with control (cont) siRNA or siRNA oligos specific for Eg5. (A and B) 48 h after siRNA transfection, the cell lysates were collected and Western blotted with anti-Eg5 and anti–α-tubulin antibodies, respectively. (B) Quantification of the expression levels of Eg5 upon RNAi. The average values of four independent experiments are shown (mean ± SD). (C) 48 h after siRNA transfection, the cells were fixed and visualized with anti–α-tubulin antibody. Bar, 5 µm. (D) 24 h after siRNA transfection, 2 mM thymidine was added to the medium, and 24 h later the cells were fixed and visualized with anti–α-tubulin antibody. Bar, 10 µm. (E and F) 20 h after siRNA transfection, the cells were transfected with a plasmid for PAUF-MycHis and 4 h later 2 mM thymidine was added to the medium. The medium and the cell lysate were Western blotted with anti-His antibody. Single and double star denote the mature and immature form of PAUF, respectively. (F) Quantification of PAUF secretion. The amount of secreted PAUF was normalized with the expression levels. The average values of four independent experiments are shown (mean ± SD). Single and double star indicate P < 0.01 and P < 0.05, respectively.