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Figure 5


Fig. 5. Overexpression of SOD2 in HeLa cells decreases autophagy and autophagic cell death induced by rotenone and TTFA. Wild-type (wt) and SOD2-overexpressing (SOD2) HeLa cells were treated with rotenone (R, 50.0 µM) or TTFA (T, 0.5 mM) as indicated. (A) ROS generation was determined following 48 hours of rotenone or TTFA treatment. DMSO is a solvent control. (B) AVO formation (i) was determined after 48 hours of treatment with rotenone or TTFA and representative pictures of HeLa (wt and SOD2) cells with GFP-LC3-labeled vacuoles (green dots, ii) were obtained by a fluorescent microscope. In HeLa (wt) cells: a, control; b, rotenone; c, TTFA. In HeLa (SOD2) cells: d, control; e, rotenone; f, TTFA. The nucleolus was stained with DAPI (blue). (C) Beclin 1 expression (i) and conversion of LC3-I to LC3-II (ii) in the presence or absence of NH4Cl (30 mM) was determined by western blot. Actin was used as a loading control. (D) Cell death was determined after cells were treated with rotenone or TTFA in the presence or absence of 3-MA (2.0 mM) and/or zVAD (0.1 mM). * Represents significant difference between rotenone or TTFA treatment alone and combined treatment with 3-MA and/or zVAD (P<0.05). @ Represents significant differences between wt and SOD2 cells treated with rotenone or TTFA (P<0.05). # Represents a lack of significant difference between rotenone or TTFA treatment alone and the combined treatment with 3-MA in SOD2 cells (P>0.05). (E) Apoptosis (formation of sub-G1 peak and TUNEL assay) was determined after treatment with rotenone, TTFA or etoposide (Et, apoptotic stimuli). # Represents significant differences between etoposide treatment and control wt cells. * Represents a lack of significant differences between wt and SOD2 cells treated with rotenone or TTFA alone (P>0.05). 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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