DNA content flow cytometric histograms of propidium iodide–stained A549 cells. DNA content flow cytometric histograms of propidium iodide–stained A549 cells. A, cantharidin dose-response. Logarithmically growing A549 cells were treated with cantharidin at the concentrations indicated for 24 h. Controls were treated with solvent alone. At the time points indicated, the cells were harvested, and DNA content flow cytometry analysis was done as described in Materials and Methods. DNA content shown (X-axis) as fluorescence, and populations of cells containing 2N and 4N DNA are shaded with dark gray (2N and 4N populations, arrowheads). S-phase cells are shaded with diagonal lines. The sub-G1 populations (dead and apoptotic cells) are shaded as light gray. B and C, FACS analysis of A549 cell cultures following release from double thymidine-induced G1 growth arrest. Double thymidine treatments were used to arrest A549 cell cultures in late G1 phase of the cell cycle. The cells were then treated with solvent (B) or cantharidin (C) and released from growth suppression. At the times indicated, the cells were fixed and processed for DNA content FACS analysis as described above. D, FACS analysis of A549 cells treated with cantharidin at timed intervals after release from growth suppression. A549 cells were arrested with double thymidine treatment and then released from growth suppression as above. Cantharidin was added to the culture at the times indicated. The cells were then fixed 16 h after they were released from double thymidine growth arrest. Data shown are representative of at least three independent experiments each conducted in triplicate. Kathy Bonness et al. Mol Cancer Ther 2006;5:2727-2736 ©2006 by American Association for Cancer Research