Figure 3.

The involvement of μ-calpain in soft substrate–induced apoptosis. siRNA was used to knockdown μ-calpain and investigated this effect on soft substrate–induced apoptosis. Normal cervical epithelial cells were transfected with 100 nM siRNA specific for μ-calpain for 48 h. Then at 48-h after transfection, the cells were harvested for analysis of protein expression by immunoblotting (A) or cultured on various conditions for 24 h and analyzed the apoptosis by PI staining (B). (C) Effects of various inhibitors on the soft substrate–induced activation of μ-calpain. PD150606 and PD151746, two specific inhibitors by blocking Ca²⁺-binding site of calpain; PD145305, a negative control for calpain inhibitors; ALLN, an inhibitor targeting catalytic binding site of calpain; Z-VAD, a pan-caspase inhibitor. Normal cervical epithelial cells were cultured on collagen gel–coated dish (control) or on collagen gel (soft substrate) for 4 h in the absence or presence of various inhibitors. Intracellular calpain activities were measured by using a fluorogenic membrane-permeable calpain substrate t-Boc-LM-CMAC. Each column represents mean ± SEM (n = 6).