Figure 5.

Cell cycle‐dependent expression of DNMT3a is regulated by Plk1‐associated kinase activity. A) DU145 and C4‐2 cells were synchronized by double thymidine block (DTB, treatment with thymidine for 16 h, release for 8 h, and a second thymidine treatment for 16 h), released into fresh medium ± 100 nm BI2536 for various times and harvested for IB and quantification of p‐DNMT3a/DNMT3a. P, phosphorylated DNMT3A; Pr, total DNMT3a. B) Immunoblot of HeLa cells treated with 100 ng mL⁻¹ nocodazole ± 50 nm BI2536 for 12 h. C) Immunoblot of DU145 cells depleted of Plk1 with shRNA. D) DU145 cells were depleted of Plk1 with shRNA, and treated with 50 ng mL⁻¹ nocodazole ± 50 nm BI2536 overnight. E) HEK293T cells were co‐transfected with Myc‐DNMT3a and increasing amounts of Flag‐Plk1. F) HEK293T cells were transfected with Plk1 (WT, T210D, or K82M) and treated with 100 nm BI2536 for 12 h. G) Immunoblot of 22Rv1 cells infected with lentivirus expressing Plk1. H) HeLa cells transfected with Myc‐DNMT3a, HA‐Ub, and Flag‐Plk1 were treated with 10 µm MG132 for 16 h and harvested for anti‐Myc immunoprecipitation (IP), followed by anti‐ubiquitin IB. I) HeLa cells were synchronized with nocodazole, incubated with 50 nm BI2536 for 24 h, and harvested for anti‐DNMT3a IP, followed by anti‐ubiquitin IB. J) HEK293T cells transfected with Myc‐DNMT3a, Flag‐Plk1, and HA‐FBW7 were harvested for IB. K) HEK293T cells transfected with Myc‐DNMT3a, HA‐FBW7, and/or Flag‐Plk1 were treated with 25 µg mL⁻¹ cycloheximide and harvested at various times for IB.