Fig. 3: RUNX2 stabilizes c-Myc via direct protein interaction and forms a complex to cooperatively activate c-Myc target gene expression.

A The impact of RUNX2 on the half-life of c-Myc in the indicated cells was evaluated by immunoblot. Relative c-Myc protein levels in the cells treated with CHX for different time points were quantified from three independent experiments. B T-47D cells with RUNX2 overexpression and MDA-MB-231 with RUNX2 knockdown were treated with 10 μM MG-132 for 4 h. c-Myc protein levels in the indicated cells were detected by immunoblot. C 293T cells were transiently co-transfected with c-Myc-HA and RUNX2-Flag or vector control for 48 h followed 4 h treatment with 10 μM MG-132. Ubiquitination levels of exogenous c-Myc in the indicated cells were detected using IP and immunoblot. D 293T cells were co-transfected with RUNX2-Flag and c-Myc-HA. Interaction between exogenous RUNX2 and c-Myc proteins was detected E T-47D cells with stable RUNX2 overexpression and MDA-MB-231 cells with stable RUNX2 knockdown, as well as the corresponding control cells were treated with 10 μM MG-132 for 4 h. Ubiquitination of endogenous c-Myc was analyzed using IP and immunoblot. F Interaction between endogenous RUNX2 and c-Myc proteins in MDA-MB-231 and 4T1 cells was detected using Co-IP and immunoblot. G His-fused c-Myc and Flag-fused RUNX2 proteins expressed in bacteria were purified and were used for the His pull-down assay. Protein interaction between the two proteins was detected using an immunoblot. Interaction of HA-tagged c-Myc with Flag-tagged full-length RUNX2 or its mutants lacking N-terminal (ΔNT), Runt-homology domain (ΔRD), or C-terminal (ΔCT) (H), as well as Flag-tagged RUNX2 with HA-tagged full-length c-Myc or its mutants lacking MB1 domain (ΔMB1), MB2 domain (ΔMB2), and MB3 domain (ΔMB3) (I) in 293T cells were detected using Co-IP and immunoblot. CHX cycloheximide, shR2 shRUNX2, IP immunoprecipitation, FL full length.