Fig. 3

Formation of the PRC1–CyclinD1–HDAC4 complex. a HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the RNF2–Cyclin D1, EZH2–Cyclin D1, HDAC4–Cyclin D1, and RNF2–HDAC4 complexes was measured by IP and IB. b HEK293 cells were transfected with HA-Cyclin D1, Myc-HDAC4, and Flag-RNF2, and the interactions between the proteins were measured by IP and IB. c, d HA-Cyclin D1, Myc-RNF2, and Myc-HDAC4 were translated in vitro and the interactions among the proteins were measured by IP and IB. e Regions of Cyclin D1 required for the interaction with RUNX3, RNF2, and HDAC4 are summarized. Cyclin D1 regions known to interact with pRB and CDK4/6 are also indicated. Cyc Box = Cyclin Box. f HEK293 cells were treated with control or HDAC4-specific siRNA (si-con or si-HDAC4), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. g HEK293 cells were treated with control, RNF2-specific, or Cyclin D1-specific siRNA (si-con, si-RNF2, or si-CycD1), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, and RNF2–BRD2 complexes was measured by IP and IB. h Schematic illustration of the process of Rpa-RX3-TR formation. Cyclin D1, which is induced 2 h after serum stimulation, interacts with PRC1 (containing RNF2) and matures into the PRC1–CyclinD1–HDAC4 complex. The PRC1–CyclinD1–HDAC4 complex then interacts with Rpa-RX3-AC to form Rpa-RX3-TR