Figure 6.

Caffeine induces apoptosis by enhancement of autophagy. (A) After PC12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMSO or MPP⁺ for 72 hours, cell viability was measured using trypan blue dye exclusion assay. Data are the means of triplicate experiments. (B) After cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMSO or MPP⁺ for 48 hours, mitochondrial membrane potential was analyzed by Jc-1 using a flow cytometry. Data are the means of triplicate experiments. (C) After PC12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMSO or MPP⁺ for 72 hours, caffeine-induced sub G₁ area was analyzed by propidium iodide staining assay using a flow cytometry. Data are the means of triplicate experiments. (D) PC12D cells were treated with H₂O or caffeine for 24 hours or staurosporine (positive control) for 3 hours and analyzed with immunoblotting for levels of caspase-3 and cleaved caspase-3. (E) After PC12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with or without 1, 3 or 5 mM 3MA for 24 hours, cell viability was measured by trypan blue dye exclusion assay. (F) PC12D cells were transfected with control siRNA or siRNAs targeting Atg7. Forty eight hours later, they were treated with 0, 1, 2.5 or 10 mM caffeine for 24 hours and mitochondrial membrane potential was analyzed using Jc-1. The knockdown effects on Atg7 were confirmed by immunoblotting using antibodies against Atg7 and actin. Data are the means of triplicate experiments. Error bars, S.D. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.