Fig. 2: BA suppresses the activity of signaling pathways and inhibits interaction of 14-3-3ζ and its client proteins in cancer cells.

a Immunoblot analysis of total and phosphorylated (p-) forms of proteins involved in cancer-associated signaling pathways in BxPC-3 cells incubated with BA (100 or 500 µM) or dimethyl sulfoxide (DMSO) vehicle for the indicated times. β-actin was examined as a loading control. b Immunoblot analysis of total and phosphorylated forms of AKT, of AKT substrates (FOXO3a, FOXO1, PRAS40, and TSC2), and of Rictor, which contributes to AKT phosphorylation, in BxPC-3 cells treated with BA as in a. c Immunoblot analysis of the phosphorylated 14-3-3 binding motif in BxPC-3 cells treated with DMSO or 500 µM BA for 24 h. d Immunoblot analysis of the seven isoforms of 14-3-3 proteins in BxPC-3 cells treated with BA as in a. e HEK293T cells transiently transfected with expression vectors for Myc epitope–tagged 14-3-3 protein isoforms (or with the empty vector) were incubated with DMSO (D) or 500 µM BA (B) for 24 h and then lysed and subjected to immunoprecipitation (IP) with antibodies to Myc. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to c-Raf. f HEK293T cells transiently transfected with an expression vector for Myc epitope–tagged 14-3-3ζ (or with the empty vector) were incubated with DMSO (D) or 500 µM BA (B) for 24 h and then lysed and subjected to immunoprecipitation with antibodies to Myc. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot analysis with antibodies to mTOR, TSC2, Raptor, Rictor, FOXO1, FOXO3a, or STAT3. g HEK293T or BxPC-3 cells were treated with DMSO (D) or 1000 µM BA (B) for 100 min, lysed, and subjected to a pull-down assay with GST-tagged 14-3-3ζ. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot analysis with antibodies to c-Raf, TSC2, Rictor, STAT3, GST, or 14-3-3ζ. h BxPC-3 cells were treated with DMSO (D) or 1000 µM BA (B) for 100 min, lysed, and subjected to a pull-down assay with GST or GST-tagged 14-3-3ζ. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot analysis with antibodies to acetyl-lysine, 14-3-3ζ, or GST. The lane labeled Recom. GST–14-3-3ζ indicates recombinant GST-14-3-3 ζ without lysate. i Schematic illustration of the proposed mechanism by which BA induces acetylation of 14-3-3 ζ, its dissociation from client proteins, and consequent dephosphorylation of the client proteins.