Figure 2. Thyme volatile oil-sensitizes TRAIL induces colon cancer cell death. (A) Human HCT116 cells (2 × 103 per well) were treated with the indicated concentrations of TS for 24 h, stained with a Live/Dead assay reagent for 30 min and then analyzed for cell cytotoxicity under a fluorescence microscope. Values below each photomicrograph represent percentage of dead cells. Data represent the means of three independent experiments (i); colon cancer cells were exposed to 0.5 pg/ml thyme volatile oil for 12 h and rinsed with PBS. Cells were then treated with 25 ng/ml TRAIL for an additional 24 h. Cell death was analyzed by the LIVE/DEAD assay (ii). Orange arrows indicates necrotic cells; pink arrows indicates live cells and white arrows indicate apoptotic cell. (B) HCT116 (5 × 102) cells seeded in 6 well plates were treated with different concentrations of TS (0-1 μg/ml), TRAIL (25 ng/ml) and TS + TRAIL for 9 days to form colonies and stained with clonogenic acid reagent to fix cells, and then incubated with crystal violet dye. Colony-forming ability was assessed by counting blue colonies. (C) TS sensitizes TRAIL-induced PARP cleavage and caspase activation in dose-dependent manner (i). Briefly, HCT116 cells (1 × 106 per well) were pretreated with vehicle control (DMSO) or indicated doses of thyme essential oil for 12 h and then rinsed, TRAIL was then added for an additional 24 h. Whole-cell lysates were subjected to Western blotting analysis using relevant antibodies. TS sensitizes TRAIL-induced PARP cleavage and caspase activation in time-dependent manner. Colon cancer cells were exposed to thyme volatile oil and TRAIL at the indicated time points (ii). Whole-cell lysates were subjected to Western blotting analysis using relevant antibodies. Used blots were stripped and reprobed with β-actin antibodies to verify equal protein loading. These are representative results of three independent experiments.