Fig. 8. Rotenone and TTFA do not induce autophagy in primary mouse astrocytes. Normal mouse astrocytes were treated with rotenone (R, 50.0 µM) or TTFA (T, 0.5 mM) over a 48-hour time course. (A) ROS generation and (B) autophagy [AVO formation and formation of GFP-LC3-labeled vacuoles (dots)] were determined as described in the Materials and Methods section. (C) Expression of beclin 1 (i) and conversion of LC3-I and LC3-II (ii) were determined by western blotting. As a positive control for conversion of LC3-I to LC3-II, astrocytes were starved of glucose and pyruvate for 3 days. Astrocytes were also incubated in the presence or absence of NH₄Cl (30 mM) and conversion of LC3-I to LC3-II was determined. (D) Cell death was determined by membrane permeabilization (i) and apoptosis was determined by the formation of sub-G1 peaks (ii). Error bars represent s.e. from three independent experiments. * Represents significant difference from control conditions (P<0.05).