FIGURE 7.

Atg5 cleavage by calpain is involved in switching ER stress-mediated autophagy to apoptosis in SPP1-depleted cells. A, MCF7 cells transfected with siControl or siSPP1 were treated with doxorubicin (1 μg/ml) for the indicated times. Equal amounts of cell lysates were analyzed by Western blotting with an anti-Atg5 antibody. Blots were reprobed with tubulin to ensure equal loading and transfer. B and C, MCF7 cells transfected with siControl or siSPP1 without or with GFP-LC3 were treated with doxorubicin in the absence (None) or presence of E64d (10 μg/ml) for 48 h, and autophagy (B) and apoptosis (C) were determined. D, MCF7 cells transfected with siSPP1 were treated with doxorubicin (1 μg/ml) for 48 h in the absence or presence of E64d (10 μg/ml), and cleavage of Atg5 was determined by Western blotting. E, MCF7 cells were transfected with siControl or siCalpain, and calpain expression was quantified by quantitative PCR and normalized to GAPDH expression. F–G, MCF7 cells transfected with siControl or siSPP1 without or with siCalpain were treated with doxorubicin for 48 h, and autophagy (F) and apoptosis (G) were determined. H, MCF7 cells transfected with siSPP1 without or with siCalpain were treated with doxorubicin for 48 h, and cleavage of Atg5 was determined by Western blotting. *, p < 0.01, **, p < 0.05. Error bars in panels B–G indicate means ± S.D.