Figure 7.

Effect of different inhibitors on LC3-II accumulation. SH-SY5Y human neuroblastoma cells were plated and allowed to adhere for a minimum of 24 h, then treated in fresh medium. Treatments were as follows: rapamycin (Rap), (A) 1 µM, 4 h or (B) 10 µM, 4 h; E-64d, final concentration 10 µg/ml from a 1 mg/ml stock in ethanol (EtOH); NH₄Cl (NH₄⁺), final concentration 10 mM from a 1 M stock in water; pepstatin A (Pst), final concentration 10 µg/ml from a 1 mg/ml stock in ethanol, or 68.6 µg/ml from a 6.86 mg/ml stock in DMSO; ethanol or DMSO, final concentration 1%. Pre-incubations in (B) were for 1 or 4 h as indicated. 10 mM NH₄Cl (or 30 µM chloroquine, not shown) were the most effective compounds for demonstrating the accumulation of LC3-II. E-64d was also effective in preventing the degradation of LC3-II, with or without a preincubation, but ammomium chloride (or chloroquine) may be more effective. Pepstatin A at 10 µg/ml with a 1 h pre-incubation was not effective at blocking degradation, whereas a 100 µM concentration with 4 h pre-incubation had a partial effect. Thus, alkalinizing compounds are more effective in blocking LC3-II degradation, and pepstatin A must be used at saturating conditions to have any noticeable effect. Images provided by C. Isidoro. Note that the band running just below LC3-I at approximately 17.5 kDa may be a processing intermediate of LC3-I; it is detectable in freshly prepared homogenates, but is less visible after the sample is subjected to a freeze-thaw cycle.