Figure 4. ACY-1215 suppresses translocation of GRP78 to the cell surface via GRP78 acetylation, which affects proliferation and apoptosis via AKT/Bad signaling. (A) The TFK-1 cells were cultured with ACY-1215 at 10 μM for 24 h. Through the biotinylation assay, we labeled and purified the GRP78 located on the plasma membrane and detected the GRP78 via western blot analysis. (B) TFK-1 cells were either untreated (Control) or treated with 10 μM ACY-1215 for 24 h. Cell surface GRP78 were measured by FACS (Pink dots, Isotype control; Blue dots, anti-GRP78 antibody). Relative cell surface-GRP78 level in either untreated (Control) or treated cells with 10 μM ACY-1215 for 24 h (n=3). (C) TFK-1 cells were either untreated (Control) or treated with 10 μM ACY-1215 for 24 h. Subsequently, the lysates from the cell pellets were immunoprecipitated with anti-GRP78 antibody and anti-acetyl-lysine antibody, respectively, and immunoblotted for acetyl-lysine and GRP78, respectively. (D) Following ACY-1215 treatment at the indicated concentrations (5, 10 and 20 μM) for 24 h, p-AKT (Ser473) levels were detected by western blot. p-AKT (Ser473) related to the proliferation of cancer cells. (E) TFK-1 cells were treated with ACY-1215 at the indicated concentrations (5, 10 and 20 μM) for 24 h. Western blot shows the levels of p-AKT (Thr308), AKT, p-Bad (Ser112), p-Bad (Ser136), Bad and B-actin. p-AKT (Thr308) related to the blockade of apoptosis via Bad phosphorylation. All the experiments were performed three times.