Figure 7.

Induction of apoptotic reaction in cells expressing mutated COMP. COS7 cells were transfected with wild-type (A) or mutated COMP (B) and cultured for 48 hours at 37°C. The cells were double-stained with a TUNEL assay kit (Roche Diagnostic, IN) (green) and an anti-COMP antibody followed by a rhodamine-labeled secondary antibody (red). Note that apoptosis occurred exclusively in cells expressing mutated COMP. Arrows show both TUNEL- and COMP-positive cells. Bar, 20 μm. C: The TUNEL- and COMP-positive cells/COMP-positive cells ratios were calculated. Apoptosis was significantly more prominent in cells expressing mutated COMP (14.6 ± 2.1%) than in those expressing wild-type COMP (4.6 ± 0.6%) (*, P < 0.005). The means ± SEM of the ratios obtained from three independent transfections are shown. D: Transfected COS7 cells were cultured for 96 hours at 37°C. As a positive control, untransfected cells were treated with 1 mmol/L STS for 12 hours at 37°C. Each genomic DNA was precipitated as described in Materials and Methods. The DNA samples were subjected to 1.5% agarose gel electrophoresis, stained with 0.1 μg/ml of Syber Green and visualized under ultraviolet light. Note that cells expressing mutated COMP exhibited DNA fragmentation, whereas those expressing wild-type COMP did not. The left lane represents 100-bp DNA size marker (Promega, WI).