Cytotoxicity assay, MRK-16 staining, rhodamine 123 efflux, and molecular response to depsipeptide of resistant HUT78 cells. HUT78 cells were selected for resistance to depsipeptide in the absence (HUT78Dp) or presence of 5 μg/mL of verapamil (HUT78DpVp). For these experiments, cells were first grown out of depsipeptide and verapamil for 2 weeks. (A) Cells were treated with depsipeptide for 3 days with or without 1 μg/mL of valspodar, an inhibitor of Pgp, and cytotoxicity assays were performed. Data are shown on a logarithmic scale. (B) The panels on the left show the results of cells stained with the Pgp-specific antibody, MRK-16 (dashed line), or with an isotype-specific antibody as a control (solid line). In the right panel, Pgp function was assayed by the ability of the cells to efflux rhodamine. Cells were first incubated with rhodamine 123 and then incubated for 1 hour in the absence of rhodamine, both steps in the absence (solid line) or presence (dashed line) of valspodar. (C) HUT78, HUT78Dp, and HUT78DpVp cells were treated overnight with the indicated amount of depsipeptide. Whole cell lysates were prepared, and 10 μg/mL of proteins were separated by PAGE. Immunoblots were performed using antibodies against acetylated histone H3, demonstrating histone deacetylase activity of the individual HDIs, and GAPDH, to confirm equivalent loading of proteins.