FIGURE 4.

Identification of p21 as a target for Ccr4d. A, the effect of Ccr4d on the expression of cell cycle-related proteins. MCF-7 cells were transfected with FLAG-Ccr4d construct or Ccr4d siRNA, and cellular lysates were prepared for Western blotting analysis with antibodies against the indicated proteins. B, expression of p53 and PUMA upon Ccr4d knockdown in MCF-7 cells. Cells were transfected with control siRNA and Ccr4d siRNA for 72 h, and cellular lysates were prepared for Western blotting analysis with antibodies against the indicated proteins. β-actin was used as a loading control. C, the expression of p21 upon Ccr4d knockdown in H1299 cells. Cells were transfected with control siRNA and Ccr4d siRNA for 72 h, and cellular lysates were prepared for Western blotting analysis with antibodies against the indicated proteins. β-actin was used as a loading control. D, Ccr4d knockdown promotes H1299 cell cycle progression. After cells were transfected with control siRNA or Ccr4d siRNA for 24 h, the cells were switched to conditioned medium without serum for another 24 h. The cells were then cultured in medium containing 10% FBS for 27 h and were collected for cell cycle analysis by flow cytometry. The data represent three independent experiments in the left panel. A representative profile is shown in the right panel. E, expression of p21 upon knockdown of Ccr4d in p53^+/+ and p53^−/− isogenic cell lines. Cells were transfected with control siRNA and Ccr4d siRNA for 72 h, and cellular lysates were prepared for Western blotting analysis with antibodies against the indicated proteins. β-actin was used as a loading control.