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Fig. S1. Production of superoxide anion and reduction of mitochondrial membrane potential induced by thymosin β4 (Tβ4) knockdown in SW480 cells are slightly reduced and increased, respectively, by vitamin C treatment.

Fig. S2. Production of superoxide anion in SW480 cells triggered by thymosin β4 (Tβ4) knockdown is not inhibited by the treatment of several commonly used anti-oxidants.

Fig. S3. Production of superoxide anion induced by thymosin β4 (Tβ4) knockdown in SW480 cells is not reduced by the addition of NOX inhibitor apocynin.

Fig. S4. Superoxide anion production from mitochondria is induced by thymosin β4 (Tβ4) knockdown in SW480 cells.

Fig. S5. Increased superoxide anion production and decreased mitochondrial membrane potential are also found in SW480 cells infected by AdTβ4sh2.

Fig. S6. Reduced protein levels of ETC components in SW480 cells are detected after thymosin β4 (Tβ4) downregulation.

Fig. S7. Increased apoptosis of SW480 cells and reduced migration ability in the SW480-derived stable clone are triggered by thymosin β4 (Tβ4) downregulation.

Fig. S8. Colocalization of mitochondria-targeted DsRed and thymosin β4 (Tβ4) is also observed in lung cancer H1299 cells as well as colon cancer HCT116 cells, and alterations in superoxide anion production and mitochondrial membrane potential triggered by Tβ4 knockdown are detected in the latter.

Data S1. Including: reagents; generation of adenovirus expressing Tβ4 shRNA2; quantitative real-time PCR; flow cytometry analyses; western blot analysis; transmission electron microscopy; determination of mtDNA levels; determination of the intracellular ATP levels; and determination of the lactate levels.

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