FIG. 4.

Involvement of caspase-3 activation in apoptosis induced by A. actinomycetemcomitans LPS in the presence of CHX. TPA-differentiated U937 cells (10⁷ cells) were treated with A. actinomycetemcomitans LPS (1 μg/ml) in the presence or absence of CHX (10 μg/ml) for 30 min, 1 h, and 3 h (A to C). Following each treatment, caspase-1 (A), caspase-3 (B), and caspase-8 (C) activities were measured using colorimetric assay kits according to the manufacturer's instructions. (D) TPA-differentiated U937 cells (10⁵ cells) were also pretreated for 1 h with a 100 μM concentration of a caspase inhibitor, Z-asp-CH2-DCB, or nothing, and were subsequently cotreated with A. actinomycetemcomitans LPS (1 μg/ml) and CHX (10 μg/ml) for 3 h. Following each treatment, the effect of Z-asp-CH2-DCB on apoptosis induced by LPS and CHX (D) was evaluated by morphological assessment using Hoechst 33258-dye, as described in Materials and Methods. Values shown (units per milligram of protein [A to C] or percent apoptotic cells [D]) are the means + SD (error bars) of three separate experiments, each conducted in triplicate. Differences from the value for untreated cells (B) or cells cotreated with LPS and CHX (D) were considered significant (**) at a P of <0.01.