Expression of genes involved in cell-cycle progression and antiapoptosis after DHMEQ treatment. (A) Immunoblot analysis of DHMEQ-treated MT-1 cells (left panels). Cells were treated with 10 μg/mL DHMEQ for 16 hours. Total cell lysates were subjected to analysis. Antibodies used for detection are presented on the left. Levels of tubulin expression are used to confirm equal amounts of total cell lysates in each lane. (Right panel) Results of densitometric analysis of detected bands. Results are expressed as relative levels compared with those of untreated samples. Tubulin bands were used to normalize densitometric measurement. (B) Activation of caspases. Cleavage of caspase-3, -8, and -9 was examined by immunoblot analysis. Samples were prepared at the indicated time points after DHMEQ treatment. Arrows indicate the position of cleaved or uncleaved fragments. (C) Inhibition of apoptosis pathways. Effects of caspase inhibitors on DHMEQ-induced apoptosis were studied using specific inhibitors for caspase-3 (Z-DEVD-FMK) and caspase-8 (Z-IETD-FMK) as well as caspase-9 inhibitor (z-LEHD-FMK). Inhibitors were added to the culture media 1 hour prior to addition of DHMEQ. (Left panel) Cell viabilities after DHMEQ treatment in the presence of specific caspase inhibitors. (Right panel) Immunoblot analysis of caspase cleavage. Protein expression levels of cleaved or uncleaved caspases are shown. UC indicates uncleaved; C, cleaved. (D) Effects of DHMEQ on cell-cycle progression. MT-1 cells were treated with DHMEQ (10 μg/mL) for indicated periods followed by PI staining and subjected to cell-cycle analysis by flow cytometry.