Figure 6

Expression of CARP-1 (A) or H89 treatment (B) causes reduced c-myc levels, in part by suppressing c-myc transcription (C). Cells were transduced with retroviruses encoding vector or CARP-1 proteins as indicated in panel A. For panel B, HBC cells were either untreated or treated with indicated doses of H89 or piceatannol. Approximately 100 μg of respective protein lysate was analyzed by SDS-PAGE, followed by western immunoblotting with either anti c-myc or topoisomerase IIα antibodies essentially as in methods. The membrane was subsequently probed with anti-actin antibody to assess loading. Presence of c-myc, topoisomerase IIα and actin proteins is denoted on the left side of each panel. In panel C, HBC cells were transfected with indicated c-myc promoter-luciferase reporter plasmid. The cells were either untreated (Control) or treated with H89. In addition, the HBC cells were separately transfected with luciferase reporter plasmids having full length c-myc promoter or its various deletions, followed by their transduction with retroviruses encoding vector or CARP-1 protein. The cell lysates were analyzed for luciferase reporter activities as in methods. Columns in the histograms represent means of two independent experiments; bars, SE. Data were analyzed using a two-tailed Student t test. For top left histogram, *P < 0.044, compared to the corresponding control, and for the lower histogram, *P < 0.0055 and **P < 0.006, compared to the respective vector-transduced controls. Panel D, Schematic of CARP-1-dependent growth suppression signaling in the presence of H89. = , Binding.