Examination of downstream events of apoptosis in VSC4.1 cells. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Western blotting to show levels of m-calpain, calpastatin, active caspase-3, ICAD (cytosolic), CAD (nuclear), and β-actin. (B) Densitometric analysis showing the m-calpain:calpastatin ratio and the ICAD (cytosolic):CAD (nuclear) ratio. (C) Determination of caspase-3 activation by western blotting and total caspase-3 activity by colorimetric assay. **P<0·01 compared to control; ##P<0·01 compared to TNF-α.