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Fig. 4. ROS scavenger tiron decreases autophagy and autophagic cell death induced by rotenone and TTFA in HEK 293 and U87 cells. Cells were treated with 50.0 µM rotenone (R), 0.5 mM TTFA (T), and/or tiron (1.0 mM). (A) ROS generation after (i) HEK 293 and (ii) U87 cells were treated with rotenone or TTFA over a 72-hour time course. (B) HEK 293 and U87 cells were treated with tiron alone or in combination with rotenone or TTTF. The percentages of (i) ROS generation, (ii) AVO formation and (iii) GFP-LC3-labeled vacuoles (dots) were determined after 48 hours. (C) Expression of beclin 1 (i) and conversion of LC3-I to LC3-II (ii) were determined by western blotting after 48 hours in the presence or absence of tiron. Actin was used as a loading control. NH₄Cl was used as a lysosomal inhibitor. (Di) Cell death was determined by membrane permeabilization following rotenone or TTFA treatment in the presence or absence of tiron in HEK 293 and U87 cells after 48 hours. (Dii) Apoptosis (formation of sub-G1 peaks) was determined in HEK 293 and U87 cells treated as above. Error bars represent s.e. from three independent experiments. P values less than 0.05 represent significant difference between conditions, as indicated.

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