JCS011163 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Histograph of acidic vacuoles in HEK 293 cells treated with rotenone or TTFA. HEK 293 cells were treated with rotenone (R, 50 mM) or TTFA (T, 0.5 mM) for 72 hours. The formation of acidic vacuoles was determined as described in Materials and methods section. The percentage is the number of events for positive staining for acidic vacuoles in the upper left and right hand quadrants.

• Supplemental Figure S2 -

  Fig. S2. HEK 293 cells induce autophagy under starvation conditions. HEK 293 cells were starved of glucose and pyruvate (G-P) for 3 days in the presence or absence of lysosomal inhibitors NH₄Cl (30 mM) or 3-MA (0.2 mM). Autophagy was determined by (A) AVO formation or (B) formation of GFP-LC3 vacuoles (dots). Standard error represents three separate experiments. P values less than 0.05 represent significant difference between conditions as indicated.

• Supplemental Figure S3 -

  Fig. S3. The percentage of apoptosis in HEK 293 and U87 cells treated with rotenone or TTFA in the presence or absence of 3-MA. Cells were treated with rotenone (R, 50 μM) or TTFA (T, 0.5 mM) in the presence or absence of 3-MA (0.2 mM). (A) The amount of apoptosis in HEK 293 cells was determined by the percentage of cells with a sub-G1 peak. (B) The amount of apoptosis in U87 cells was determined by both sub-G1 peak and TUNEL analysis as described in Materials and methods section. Standard error represents three independent experiments. P values less than 0.05 represent significant difference between conditions as indicated.

• Supplemental Figure S4 -

  Fig. S4. Affects of siRNAs against the autophagic genes beclin 1 and ATG5 on rotenone- or TTFA-induced autophagy, cell death, apoptosis and ROS generation in HEK 293 cells. HEK 293 cells were not transfected or were transfected with scrambled siRNA and siRNAs against beclin 1 and ATG5. (A) Cells were lysed and western blotted for beclin 1 and ATG-5. Blots were stripped and re-probed with anti-actin antibody for equal loading. (B) AVO formation, (C) formation of GFP-LC3 vacuoles (dots), (D) cell death and (E) apoptosis (formation of sub-G1 peaks) were determined as described in Materials and methods section. Standard error represents three independent experiments. * represents significant differences between siRNA and non-siRNA conditions.

• Supplemental Figure S5 -

  Fig. S5. Histograph of ROS generation in HEK 293 cells following TTFA treatment. HEK 293 cells were treated with TTFA (T, 0.5 mM) for 24 hours. The amount of ROS generation was determined as described in Materials and methods section. The percentage of ROS generation is denoted as a percentage within the gated populations (M1).

• Supplemental Figure S6 -

  Fig. S6. ROS scavengers prevent TTFA-induced ROS, autophagy and cell death in HEK 293 cells. Cells were treated with TTFA (T, 0.5 mM) for 48 hours in the presence or absence of L-cysteine (Cys, 10.0 mM) or glutathione (GSH, 10.0 mM). The amount of (A) ROS generation, (B) AVO formation, (C) formation of GFP-LC3 vacuoles (dots), and (D) cell death was determined as described in Material and methods section. Standard error represents three independent experiments. P values less than 0.05 represent significant difference between conditions as indicated.

• Supplemental Figure S7 -

  Fig. S7. Expression of SOD2 in HEK 293, U87, mouse astrocytes and HeLa cells. Cells were lysed and western blotted for SOD2 expression. Blots were stripped and re-probed for actin as a loading control. HeLa cells overexpressing SOD2 were lysed and western blotted to compare SOD2 levels to wild-type HeLa cells.

• Supplemental Figure S9 -

  Fig. S9. Silencing SOD2 expression by siRNA increases autophagy and autophagic cell death induced by TTFA in HEK 293 cells. HEK 293 cells were not transfected or were transfected with scrambled and SOD2 siRNAs. (A) Cells were lysed and western blotted for SOD2. The blot was stripped and re-probed for actin. After HEK 293 cells were treated with TTFA (T, 0.5 mM), (B) ROS generation following treatment of 6 hours and AVOs formation following treatment of 24 hours, and (C) cell death following treatment of 24 hours in the presence and absence of 3-MA (2.0 mM) were determined as described in Materials and methods section. Standard error represents three independent experiments.

• Supplemental Figure S10 -

  Fig. S10. Knockdown of beclin 1 and ATG5 fails to reduce rotenone- or TTFA-induced ROS generation in U87 cells. U87 cells were untreated or transfected with scrambled siRNA and siRNAs against beclin 1 and ATG5. The cells were then treated with rotenone (R, 50 mM) or TTFA (T, 0.5 mM). The amount of ROS generation was determined as described in Material and methods section. Standard error represents three independent experiments. * represent a lack of significant difference from control conditions (P>0.05).

• Supplemental Figure S11 -

  Fig. S11. Mouse primary astrocytes under starvation conditions undergo autophagy. Mouse primary astrocytes were starved of glucose and pyruvate for 72 hours. Astrocytes were also incubated in the presence or absence of NH₄Cl (30 mM) and 3-MA (0.2 mM). The amount of (A) AVO formation and (B) formation of GFP-LC3 vacuoles (dots) were determined as described in Materials and methods section. Standard error represents three independent experiments. P values less than 0.05 represent significant difference between conditions as indicated.