Fig. 5
Multiple signals contribute to the R-point transition. a HEK293 cells were treated with MEK1 inhibitor (U0126, 1 μM). Time-dependent interactions of BRD2–RUNX3 and p300–RUNX3, as well as phosphorylation of ERK1/2, were monitored by IP and/or IB. b HEK293 cells were treated with control or CyclinD1-specific siRNA (si-con or si-CycD1). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 and pRB at Ser-795 were measured by IP and IB. c Time-dependent formation of the JNK-CDK4 complexes and phosphorylations of RUNX3 at Ser-356 and CDK4 at Thr-172 were measured by IP and IB. d HEK293 cells were treated with JNK inhibitor (JNK-IN-8, 1 μM). Time-dependent formation of the BRD2–RUNX3 and RUNX3–CDK4 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. e HEK293 cells were treated with control or JNK-specific siRNA (si-con or si-JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. f HEK293 cells were transfected with Myc-RUNX3 and Flag-CDK4 WT or Flag-CDK4-T172A (CDK4 mutant defective in phosphorylation by JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent phosphorylation of pRB and expression of ARF were measured by IB. g HEK293 cells were treated with control or PIK3CA-specific siRNA (si-con or si- PIK3CA). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. h HEK293 cells were treated with control or mTORC1 inhibitor (Rapamycin, 100 nM). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. Ribosomal protein S6 kinase beta-1 (S6K1), which is phosphorylated by mTOR signaling, was used for control
