TPA-induced activation of caspases is not related to apoptosis.

U937 cells were treated with 20 nM TPA for 24 hours (A,B) or for indicated times (C,D) or with 50 μM etoposide for 4.5 hours (VP16). Fluorescence microscopy analysis of caspase-3 (A) or caspase-9 (B) active fragments in U937 cells. Cleaved caspase-3 and caspase-9 were assigned the color green, and nuclei labeled with Hoechst were stained in blue. (C, upper panel) Agarose gel electrophoresis of DNA. The percentage of apoptotic cells, determined by fluorescence microscopy after Hoechst staining, is indicated above (mean of triplicates; SD less than 1%). (C, lower panels) Western blot analysis of poly(ADP-ribose)polymerase (PARP) and acinus in whole-cell extracts. HSC70 is the loading control. Numbers on the right indicate molecular weight in kilodaltons; * indicates cleavage products. (D) The percentages of cells labeled with annexin V–FITC (left panels) and propidium iodide (PI; right panels) were measured by flow cytometry. One representative of at least 3 independent experiments is shown.