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PCR was then carried out using the sense primer 5��-AAACAGAGTAGCAGCTCAGACTGC-3�� and antisense primer 5��-TCCTTCTGGGTAGACCTCTGGGAG-3��, encompassing http://www.selleckchem.com/products/PLX-4032.html the spliced sequences of human XBP1. The fragments were further digested by Pst I at 37 ��C for 1 h, and then they were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. All assays were conducted in three independent experiments. In cell-survival assay and luciferase activity assay, results are expressed as the mean value �� standard deviation (SD). In luciferase activity assay, statistical analysis was performed using GraphPad Prism software. The statistical significance of differences between two groups was analyzed with two-sided unpaired Student's t�Ctests (*P http://www.selleck.cn/products/U0126.html on induction of apoptosis by Annexin V-PE /7-AAD staining and flow cytometry analysis. As shown in Fig. 1C, HDMC effectively induced apoptosis in H157 and H460 cells. In addition to induction of apoptosis, HDMC also induced cell-cycle arrest at the G2/M phase in H157 and H1792 cells when treated with high concentration (20 ��mol L?1) (Supporting Information Fig. S1). Collectively, these results show that HDMC inhibits the growth of human lung cancer cells through induction of apoptosis and/or cell-cycle arrest. As caspase cascade activation is an important marker of apoptosis, we further examined whether HDMC treatment could induce caspase activation by Western blot assay. The results showed that in NSCLC cells H460, H157 and H1792, HDMC treatment decreased the levels of the proforms of caspases 8, 9 and 3, and increased their cleaved forms as well as http://www.selleckchem.com/products/INCB18424.html cleavage of PARP, the substrate of activated caspase 3. Moreover, the caspase activation was dependent on HDMC concentrations (Fig. 2A). We also demonstrated that in cell lines H460 and H157, HDMC activated caspase cascades in a time-dependent manner (Fig. 2B). To further examine the role of caspase cascades in HDMC's function, we took use of pan-caspase inhibitor Z-VAD and found that Z-VAD dramatically inhibited HDMC-induced caspase activation (Fig. 2C) and cell death (Fig. 2D). Taken together, our studies indicate that HDMC induces caspase-mediated apoptosis in NSCLC cells. DR5 is an initial inducer and plays a crucial role in the extrinsic apoptotic pathway.