Figure 2.

Caffeine does not block autophagosome-lysosome fusion. (A–D) SH-SY5Y (A) or heLa (C) cells treated with 10 mM caffeine with or without E64d (10 µg/ml) and pepstatin A (10 µg/ml) were analyzed by immunoblotting with antibodies against LC3 and actin. Densitometry analysis of LC3 levels relative to actin in SH-SY5Y (B) and HeLa (D) cells was performed using three independent experiments. (E and F) HeLa cells treated with various concentrations of caffeine for 48 hours were analyzed using confocal microscopy (E). Number of the autolysosomes and autophagosomes were automatically counted using imageJ “Colocalization” Plugin and the ratios were calculated (F).