Fig 2: CDK4 plays key roles in the R-point transition. a HEK293 cells were serum-starved for 24 h, stimulated with 10% serum, and harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3, E2F1–RUNX3, CDK4–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, p16INK4a–CDK4, p21–CDK4, Cyclin D1–CDK4, and HDAC4–CDK4 complexes was measured by IP and IB. Time-dependent phosphorylation of pRB (at Ser-795) and ERK1/2 was measured by IB. b PLA assay showing the RUNX3–CDK interaction 2 h after serum stimulation. c HEK293 cells were treated with control or CDK4-specific siRNA (si-con or si-CDK4), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, CDK4–RUNX3, HDAC4–RUNX3, and Cyclin D1–RUNX3 complexes and phosphorylated RUNX3 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. d HEK293 cells were treated with CDK4 inhibitor (PD0332991, 500 nM), 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. Time-dependent expression of ARF was measured by IB. e Cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent RUNX3–CDK4 interaction and RUNX3 phosphorylation at Ser-356 were measured by IP and IB. f, g HEK293 cells were transfected with Myc-RUNX3, Myc-RUNX3-S356A, or Myc-RUNX3-S356E, serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3 complex, RUNX3 phosphorylation at Ser-356, and ARF expression were monitored by IP and IB. h Schematic illustration of the process of Rpa-RX3-AC ? Rpa-RX3-TR transition. CDK4 of Rpa-RX3-AC and Cyclin D1 of PRC1–Cyclin D1–HDAC4 provide docking sites for the interaction of the two complexes, enabling formation of Rpa-RX3-TR

Fig 3: The active pRb signalling pathway mediates HIS.(a) mRNA levels of p14/ARF, p15/INK4b, p18/INK4c and p19/INK4d in Ag WT and HIS BRCA1mut/+ HMECs. The values were determined using qRT–PCR and normalized to proliferating cells (represented by line set at 1). (b) Western blot analysis of phospho-pRb (Ser795), total pRb and Cyclin A levels in Prolif and Ag/HIS WT and BRCA1mut/+ HMECs and Prolif and M0/HIS WT and BRCA1mut/+ HDEs. Bar graphs bellow the blots show the average levels of phospho-pRb (Ser795), total pRb and Cyclin A levels determined using western blot analysis in Ag WT (n=3) and HIS BRCA1mut/+ (n=3) HMECs and M0 WT (n=3) and HIS BRCA1mut/+ (n=3) HDEs. (c) pRb knockdown in proliferating BRCA1mut/+ HMECs (mRNA levels) and growth curve of control (shScr) and shpRb BRCA1mut/+ HMECs. (d) Representative images and summary table of significant genetic and chromosomal events determined by karyotype analysis in proliferating shScr (control) and shpRb BRCA1mut/+ HMECs. (e) Western blot analysis of pRb, p53 (Ser15), total p53, p21 and p27 levels in growth-arrested shScr (control) and shpRb BRCA1mut/+ HMECs. Student's two-tailed t- and ?2-tests were used to calculate P values. (*) indicates P value within the 0.05 level of significance. Error bar, s.e.

Fig 4: Pin1–Rb interaction is induced by G1-S Cyclin-mediated phosphorylation. (a) U2-OS cells were treated with vehicle (dimethyl sulfoxide, DMSO) or 30 µM roscovitine for 18 h. Cell lysates (500 µg total protein) were pulled down by GST-Pin1, followed by western blotting for Rb. Total protein (30 µg) was loaded as an input control. Hyper-pRb, hyperphosphorylated Rb; Hypo-pRb, hypophosphorylated Rb. (b) Diagram of RbLP highlighting seven pS/T-P putative Pin1-binding sites within the Rb C-pocket (RbC), as well as two double-point mutations to Ala (S807A;S811A, denominated 2S, and T821A;T826A, denominated 2T) and mutation of all Ser or Thr residues to Ala residues (7A) used for RbC-Pin1-binding studies. (c) U2-OS cells were transfected with WT or mutant RbLP. Cell lysates were subjected to GST-Pin1 pull-down assay and western blotted for Rb to assess binding. (d) Saos-2 cells were co-transfected with Rb along with Cyclin A, Cyclin B1, Cyclin D1, Cyclin E or a vector control, as shown. Cell lysates were subjected to GST-Pin1 pull down followed by western blotting for Rb

Fig 5: 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