FIGURE 5.

TWEAK-Fn14 signaling is required for mechanical stretch-induced cell death. A, MC3T3-E1 cells were stimulated with cyclic mechanical stretch (12%) for 6 h. After stretch stimulation, cells were incubated for the indicated periods. The cells were then lysed and analyzed by immunoblotting (IB) using the indicated antibodies. B, MC3T3-E1 cells were stimulated with cyclic mechanical stretch (12%) for 6 h. After stretch stimulation, cells were incubated for 12 h in the presence or absence of H₂O₂ with the indicated concentrations. The cells were then lysed and analyzed by immunoblotting using the indicated antibodies. C, MC3T3-E1 cells were transfected with the indicated expression plasmids. After 24 h, cells were lysed and analyzed by immunoblotting using the indicated antibodies. D, MC3T3-E1 cells were transfected with siRNA as a negative control or with siRNA for TWEAK (TWEAK #1 and TWEAK1 #2). After 72 h, the cells were subjected to cyclic stretch stimulation (12%) for 6 h, and then the cells were incubated for 12 h. The cell lysates were subjected to immunoblotting using the indicated antibodies. E, quantitative RT-PCR of TWEAK under the same conditions as in D. Error bars indicate mean ± S.E. *, p < 0.05, Student's t test. F, MC3T3-E1 cells were stimulated with cyclic mechanical stretch (12%) for 6 h. After stretch stimulation, cells were incubated for 12 h. The cells were then subjected to immunostaining with anti-active caspase 3 and TUNEL staining. A–D and F, the data represent one of at least three experiments. DIC, differential interference contrast.