Fig. 8.

SIRT1 regulates ubiquitination and activity of c-MYC. (A) HCT-116 cells were transiently transfected with wild-type c-MYC together with the indicated HA-tagged ubiquitin constructs. Forty hours after transfection, cells were treated with DMSO or the SIRT1 inhibitor tenovin-6 (10 μM) for 8 h followed by immunoprecipitation of c-MYC and immunoblot detection of HA-ubiquitin using a monoclonal HA-specific antibody (12CA5). Immunoprecipitated c-MYC was detected using a monoclonal c-MYC–specific antibody (C33). (B) Densitometric analysis of c-MYC ubiquitination. As in A, HCT-116 cells coexpressing wild-type c-MYC or the Myc-K6R were cotransfected with wild-type ubiquitin or mutant ubiquitin (K48R-Ub, K63R-Ub) constructs. The signal intensities of the blots were quantitated with a CCD camera. (C) HEK-293 cells were cotransfected with the M4–min-tk–luc reporter construct (38) with four MYC/MAX binding sites and expression plasmids encoding c-MYC, c-MYC-K323R, HA-SIRT1, and HA-SIRT1-H363Y. Mean values of three independent experiments performed in duplicate are shown. Western blot analysis of the indicated proteins detected in reporter assay lysates is shown in the lower panel. (D) qPCR analysis of the c-MYC target gene DKC 11 h after activation of a tetracycline-regulatable c-MYC allele in P493-6 B cells stably expressing pINCO-SIRT1 or the vector backbone pINCO as a control. Error bars represent SD of biological triplicates.