Cdc27 phosphorylation sensitizes tumor cells to curcumin. A, Immunoblot of Cdc27 in various cell lines, DAOY, NT2, D283 Med, D341 Med (brain); HCT116 (colon); HT1376, RT4 (bladder); MDCK (kidney). The arrow indicates the band corresponding to phosphorylated Cdc27 and arrowhead shows unphosphorylated Cdc27. Actin immunoblot is shown as loading control. B, Cdc27 immunoblot of cell lines in (A) after treatment with 20 μM curcumin for indicated times. Arrows indicate crosslinked Cdc27, while arrowheads show phosphorylated Cdc27. Equal amounts of protein were used as shown by actin immunoblot. C, Cytotoxicity of curcumin in six different cell lines with different Cdc27 phosphorylation levels. LDH release was determined after 24 h of exposure to curcumin at indicated concentrations. Data are the mean ± SEM of three independent experiments. D, Immunoblots of cleaved PARP and caspase-3 as indicators of apoptosis upon exposure to 20 μM curcumin. Tubulin immunoblot ensures equal amounts of protein being used for analysis. Arrows indicate cleaved PARP.