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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Thymosin β4 (Tβ4), overexpressed in various tumors, has been shown to be involved in cellular anti-oxidation. Reactive oxygen species (ROS) function as signaling molecules and play certain roles in tumor progression. To assess the anti-oxidative role of endogenous Tβ4 in tumor cells, its expression in SW480 cells was knocked down by a shRNA, which induced significant increases of ROS. Interestingly, some cristae-lost and several electron-dense mitochondria appeared in cells with Tβ4 knockdown that was accompanied by a marked decline of the membrane potential of these organelles. Strikingly, while the ATP and lactate levels in SW480 cells were notably elevated by Tβ4 downregulation, this treatment significantly diminished the mitochondrial DNA copy number and protein levels of several subunits of the electron transport complexes. Finally, immunofluorescent staining results suggested the presence of Tβ4 in mitochondria. To the best of our knowledge, this is the first report to demonstrate that Tβ4 knockdown can disrupt the morphology and some crucial functions of mitochondria in human colorectal carcinoma (CRC) cells. (Cancer Sci 2011; 102: 1665–1672)

Thymosin β4 (Tβ4), isolated originally from calf thymus(1) and subsequently found in a variety of tissues including tumors,(2) is the main G-actin sequestering peptide in mammalian cells.(3) In addition, this protein also participates in a number of physiological(4) and even some pathological processes.(5) Among a variety of its physiological effects, Tβ4 is well known for its anti-oxidative activities because this peptide not only can be oxidized to sulfoxide (as a reactive oxygen species [ROS] scavenger) both in vitro and in cells,(3,6) but also protects human corneal epithelial cells from oxidative damage indirectly.(7)

Mitochondrion, a double-membraned organelle, contains a circular genome and forms five membrane-embedded multi-subunit complexes that constitute the oxidative phosphorylation (OXPHOS) system for ATP generation.(8) In addition, other than xanthine oxidase (XO), cytochrome P450 monooxygenases, uncoupled nitric oxide synthase (NOS) and NADPH oxidases (NOX),(9) mitochondrion is the major source of reactive oxygen species (ROS). In fact, metabolically produced ROS can come from the mitochondria as a by-product of electron transport,(10) and this organelle constantly releases approximately 1–3% of its electrons as ROS from the electron transport complex (ETC), especially complexes I and III.(11) Besides functioning as signaling molecules, ROS plays certain roles in tumor progression.(12)

4 overexpression and ROS have been proven to contribute to the malignant progression of various tumors; hence, the anti-oxidative role of endogenous Tβ4 in tumor cells was analyzed in the present study. To achieve this goal, two recombinant adenoviruses carrying DNA fragments encoding Tβ4 shRNA were generated and used to infect SW480 human colon cancer cells. Surprisingly, our findings suggest that this G-actin binding protein might play a role in maintaining the redox state by regulating the integrity of mitochondria in SW480 cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell culture.  SW480 human colorectal carcinoma cells were cultured in Leiboitz’s L-15 medium (Invitrogen, Camarillo, CA, USA) containing 10% fetal calf serum (FCS), 1% penicillin-streptomycin-amphotericin (PSA) and 1%l-glutamine in a 37°C incubator without CO2.

Generation of adenoviruses expressing Tβ4 shRNA and its scramble control.  A 68-base oligonucleotide 5′-GATCCCCCTGAGATCGAGAAATTCGATAAGTTCAAGAGACTTATCGAATTTCTCGATCTCAGTTTTTA-3′ (hTβ4sh1)(13) that included the targeting sequence (underlined) along with the flanking sequence was annealed to its complementary strand and cloned into the pSuperior-puro (Oligoengine, Madison, WI, USA). Another oligonucleotide, 5′-GATCCCCGGATCAAGTAATGATCGAAGCATTTCAAGAGAATGCTTCGATCATTACTTGATCCTTTTTA-3′ (hTβ4scr), after being annealed to its complementary strand, was ligated with a similar vector. The DNA fragments containing these sequences together with the H1 promoter obtained by PCR amplification were then subcloned into the pShuttle vector (Stratagene, La Jolla, CA, USA) to generate pShuttle-H1-hTβ4sh1 and pShuttle-H1-hTβ4scr, respectively. The procedures for generating recombinant viruses and producing them in large quantity were carried out primarily as previously described.(14)

Immunofluorescent and MitoTracker staining.  SW480 cells were seeded onto glass coverslips placed previously in six-well plates. After attachment, cells were infected by viruses for different periods before being fixed with 4% paraformaldehyde (in PBS). After permealization with 0.5% Triton X-100 and blocking with 2% FCS, cells were incubated with a polyclonal antibody against Tβ4 (1:200) or incubated simultaneously with an anti-Tβ4 antibody and antibodies against various mitochondrial components (voltage-dependent anion channel [VDAC], succinate dehydrogenase B [SDHB], heat shock protein 60 [HSP60] and mitochondrial transcription factor A [mtTFA]) overnight at 4°C. Cells were then washed with PBS before the addition of a FITC-conjugated goat anti-rabbit IgG (3 μg/mL) and/or a rhodamine-conjugated goat anti-mouse IgG (3 μg/mL). After sitting at 37°C for 30 min, cells were washed and subsequently incubated with DAPI (1 μg/mL) for 10 min. The coverslips were mounted onto slides with DAKO (Glostrup, Denmark) fluorescent mounting medium (S3023). Fluorescent images were visualized by a confocal microscope (Olympus FV1000, Olympus Corp., Tokyo, Japan).

For double staining of MitoTracker Orange (Invitrogen) and Tβ4, medium was replaced by fresh medium containing 1 μM MitoTracker Orange and incubated for 30 min before cells were fixed for further immunostaining as described above. For examination of the colocalization of mitoDsRed signals and Tβ4, cells were transfected with a plasmid encoding mitoDsRed and 48 h later cells they were processed for Tβ4 immunostaining.

For fission determination by MitoTracker Orange, fluorescent images were visualized by a confocal microscope (Olympus FV1000). The criteria for fusion and fission are >70% mitochondria that present as a linear or fragmented phenotype, respectively, while intermediate shows less than 70% linear or fragmented phenotype.

Statistical analysis.  Differences between SW480 cells infected by AdTβ4sh and AdTβ4scr with various parameters (different time points and drug treatment) analyzed were assessed by one-way anova. All statistical analyses were performed using the SPSS software system (version 14.0, IBM Corp., Armonk, NY, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

4 knockdown stimulates the production of ROS in SW480 cells. 4 is overexpressed in various tumor cells; however, little is known about the anti-oxidative role of endogenous Tβ4 in human colon cancer cells. Hence, we tried to elucidate the role of this abundant peptide in defending against intracellular oxidative stress. After effective downregulation of both mRNA and protein levels of Tβ4 by its shRNA expressed from a recombinant adenovirus was demonstrated in SW480 human colon cancer cells (Fig. 1), not only were the intracellular levels of two main ROS measured but also the effects of several commonly used antioxidants were examined. As expected, marked increases in both superoxide anion and hydrogen peroxide were observed in these cells after Tβ4 knockdown (Fig. 2a,b) and the former were only slightly reduced by vitamin C treatment (Fig. S1). In contrast, elevation of ROS in SW480 cells triggered by Tβ4 knockdown was not inhibited by the treatment of antioxidants including N-acetylcysteine (NAC), Trolox, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP, a SOD mimetic) and catalase (Fig. S2), suggesting that extensive and sustained oxidative stress is induced by Tβ4 knockdown in these cells. Interestingly, this elevation of superoxide anion production was not blocked by the treatment of apocynin, a specific inhibitor of NOX (Fig. S3), suggesting that mitochondrion is the likely source of ROS. In support of this speculation, a significant increase of superoxide anion was also detected in mitochondria of SW480 cells after being infected by AdTβ4sh1 using a mitochondrial-specific detecting probe (MitoSOX, Invitrogen) (Fig. 2c, Fig. S4). As mitochondrion appeared to be the main source of superoxide anion in Tβ4-downregulating SW480 cells, we then used transmission electron microscopy (TEM, Joel Ltd., Tokyo, Japan) to examine whether the morphology of this organelle was also affected by such treatment. As shown in Figure 2d, some electron-dense and several cristae-lost mitochondria were found in these cells, suggesting degeneration of these organelles.

image

Figure 1.  RNA and protein levels of thymosin β4 (Tβ4) in SW480 cells are effectively downregulated by its shRNA. (a) Cells were infected with AdTβ4scr or AdTβ4sh1 at MOI 200 and total RNA were isolated at different time points after infection. Reverse transcription was then carried out using 5 μg total RNA as templates and aliquots of cDNA were subjected to real-time PCR amplification in a Bio-Rad MJ Mini thermocycler (Flintshire, UK). Data are shown as the mean of at least three determinations ± SD. P-values <0.01 are annotated by ** when compared with AdTβ4scr-infected cells by one-way anova. (b) Cells were treated as above for 24 and 48 h before being fixed with 4% paraformaldehyde. The protein levels of Tβ4 in these cells were then analyzed by immunofluorescence staining. Photographs were taken by an Olympus FV1000 confocal microscope (×100).

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image

Figure 2.  Reactive oxygen species (ROS) production from mitochondria is induced by thymosin β4 (Tβ4) knockdown in SW480 cells. Cells infected with AdTβ4scr or AdTβ4sh1 for the indicated times were collected and stained with (a) 5 μg/mL hydroethidine (HE) and (b) 1 mM dichlorofluorescein diacetate (DCFH-DA) at 37°C for 30 min before the fluorescence of cells being determined by flow cytometry. Data are shown as the mean of at least three determinations ± SD. P-values <0.05 and <0.01 are annotated by * and **, respectively, when compared with AdTβ4scr-infected cells by one-way anova. (c) Cells treated as above for the indicated periods were incubated with 5 μΜ mitoSOX (Invitrogen) at 37°C for 30 min and their fluorescence was subsequently analyzed by flow cytometry. (d) Cells were infected with AdTβ4scr (left panel) and AdTβ4sh1 (right panel) for 48 h and the attached cells were collected for transmission electron microscopy as described in the Data S1. Arrows and asterisks indicate the electron-dense and cristae-lost mitochondria, respectively. Scale bars, 1 μm.

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Mitochondrial functions are altered by Tβ4 downregulation.  As a drastic increase of ROS levels and abnormal mitochondrial morphology were found in Tβ4-knockdowned SW480 cells, the presence of some dysfunctional mitochondria was consequentially postulated. To verify this speculation, both immunofluorescent microscopy and flow cytometry were applied to further analyze the mitochondrial membrane potential in normal and Tβ4-downregulating SW480 cells after they were stained with MitoTracker Orange and by JC-1, respectively. As expected, a drastic reduction of mitochondrial membrane potential was detected in these cells after Tβ4 knockdown (Fig. 3a,b). To exclude the off-target effect of shRNA, another shRNA targeting the 3′ end of Tβ4 mRNA (Tβ4sh2, generated as described in Data S1) was used and similar phenomena (increase in superoxide anion and decrease of mitochondrial membrane potential) were also observed (Fig. S5). These results clearly indicate that Tβ4 knockdown could reduce the mitochondrial membrane potential in SW480 cells.

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Figure 3.  Mitochondrial dysfunction is induced by thymosin β4 (Tβ4) knockdown in SW480 cells. (a) Cells infected with AdTβ4scr or AdTβ4sh1 for 24 and 48 h were stained with cell permeable MitoTracker Orange for 1 h before being fixed with 4% paraformaldehyde, followed by incubation with DAPI. Photographs were taken by an Olympus FV1000 confocal microscope (×100). Cells treated as above for the indicated times were stained with vital mitochondrial dye JC-1 (1.25 μg/mL) at 37°C for 10 min before the fluorescence was determined by flow cytometry. The status of the mitochondrial membrane potential was represented by the red/green ratio. (c–g) Cells treated as above for 24, 48 and 72 h were harvested and total lysates were prepared for western blot analysis using antibodies against NDUFS3 (mitochondrial ETC. I), succinate dehydrogenase B (SDHB) (II), UQCRC2 (III), COX II (IV) and ATP5β (V), respectively. The bar graphs shown represent the means of three determinations ± SD. (h) Cells treated as above for the indicated times were harvested. For estimating the mitochondrial DNA copy number, genomic DNA extracted from the aforementioned samples was subjected to real-time PCR analysis as described in the Data S1. Data are shown as the mean of at least three determinations ± SD. P-values <0.05 and <0.01 are annotated by * and **, respectively, when compared with AdTβ4scr-infected cells by one-way anova.

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It is conceivable that inhibition of the respiratory chain could increase superoxide anion production in mitochondria(15–17) and such ROS has the potential to cause protein oxidation, lipid peroxidation and DNA damage.(18) As an increase of superoxide anion was found in SW480 cells after Tβ4 knockdown, western blotting was performed as described in Data S1 to assess whether the expression levels of various subunits in the mitochondrial electron transport complexes (mETC) were also affected by this treatment. In contrast to the notion that mitochondria-generated ROS function as a regulatory signal allowing adjustment of the cellular OXPHOS capacity,(19) we found a marked reduction of the subunits in OXPHOS complexes (except that of complex III) in Tβ4-downregulating SW480 cells (Fig. 3c–g, Fig. S6). Collectively, although the precise mechanisms for the above-mentioned mitochondrial changes in Tβ4-knockdowned SW480 cells are not clear, excessive ROS produced by the defective mitochondria that cause oxidative damages to cellular constituents, especially those in the mitochondria, is postulated.(20)

We next analyzed whether the amount of mitochondrial DNA (mtDNA) was increased by this treatment because the mitochondrial DNA copy number has been shown to be elevated by oxidative stress.(21,22) To our great surprise, the amount of mtDNA in SW480 cells was dramatically reduced by Tβ4 knockdown (Fig. 3h).

Elevated ATP levels induced by Tβ4 knockdown in SW480 cells are due to increased glycolysis.  Although decreased mitochondrial membrane potential and protein levels of various components of the ETC were detected in Tβ4-knockdowned SW480 cells, a modest but nonetheless significant increase of ATP (Fig. 4a) and a higher level of lactate (Fig. 4b) were found in these cells, implying elevated aerobic glycolysis. Intriguingly, both ATP and lactate levels in Tβ4-downregulating cells were back to normal in the presence of oxamate, an inhibitor of lactate dehydrogenase (LDH) (Fig. 4c,d), suggesting that glycolysis might be activated simultaneously in these cells to compensate the impaired ATP synthesis resulting from defective mitochondria. Some of the aforementioned scenarios were also observed in patients with various mitochondrial diseases.(23,24)

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Figure 4.  Elevation of both ATP and lactate levels are induced by thymosin β4 (Tβ4) knockdown in SW480 cells. (a) Cells infected with AdTβ4scr or AdTβ4sh1 for the indicated times were harvested and their ATP levels were measured as described in the Data S1. (b) Culture media were collected and their lactate levels measured as described in the Data S1. (c,d) Cells infected with AdTβ4scr or AdTβ4sh1 and treated with or without 50 mM oxamate for 48 h before their extracellular lactate and intracellular ATP levels were measured as described above. #P < 0.01 and **P < 0.01 when compared with Mock and AdTβ4scr-infected cells, respectively, by one-way anova.

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4 is present in the mitochondria of SW480 cells.  Since mitochondrial function(s) was drastically affected by Tβ4 knockdown, we postulated that Tβ4 might not only play some crucial roles in mitochondria but also be present in this organelle. Therefore, localization of Tβ4 in the mitochondria of SW480 cells was examined by fluorescent microscopy after cells were co-stained with MitoTracker Orange and anti-Tβ4 antibody or stained with a similar antibody and together with antibodies against other mitochondrial matrix markers such as mtTFA and HSP60. Additionally, SW480 cells were also stained with anti-Tβ4 antibody after they were transiently transfected with a plasmid that encodes a fluorescent protein DsRed carrying a mitochondrial matrix targeting sequence (pDsRed2-mito; Clontech, Mountain View, CA, USA).(25,26) While a strong colocalization was found between Tβ4 and mitochondrial matrix markers (mtTFA and HSP60) as well as DsRed, little co-existence of this G-actin binding peptide with other mitochondrial proteins (e.g. VDAC and SDHB, which are localized at OMM and IMM, respectively) was observed in these cells (Fig. 5a). Finally, elevated fission of mitochondria was also detected in SW480 cells after Tβ4 knockdown (Fig. 5b), which might be attributed to a reduction in mtDNA because mtDNA-depleted dysfunctional mitochondria are small and rounded, suggesting that these organelles are undergoing f ission.(27)

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Figure 5.  Colocalization of mitochondria-targeted DsRed and thymosin β4 (Tβ4) is found and Tβ4 downregulation induces a dramatic increase of mitochondrial fission in SW480 cells. (a) SW480 cells were stained with MitoTracker Orange for 1 h or transfected with pDsRed2-mito for 48 h before being fixed with 4% paraformaldehyde, followed by incubation with an anti-Tβ4 antibody and then a FITC-labeled goat anti-rabbit IgG and DAPI. SW480 cells were fixed with 4% paraformaldehyde, followed by the procedures described above except for being incubated simultaneously with an anti-Tβ4 antibody and antibodies against VDAC, SDHB, HSP60 and mtTFA. Photographs were taken by an Olympus FV1000 confocal microscope (×100). (b) Cells infected with AdTβ4scr or AdTβ4sh1 for the indicated times were harvested and stained with 1 μM MitoTracker Orange. Fluorescent images were visualized using a confocal microscope (Olympus FV1000) at a magnification of ×100. Typical images of mitochondrial morphology are shown on the right and the percentage of fusion, fission and their intermediate were calculated by dividing the number of each population with that of the cells examined (100–150) and represented by bar charts in (b). Scale bar, 10 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Our previous study found that Tβ4 overexpression is associated with the malignancy of colon cancer(28) and work from others has suggested that ROS contributes to the carcinogenesis and malignant progression of tumor cells.(12,29) In addition, a recent report showed that drug resistance, proliferation and anti-apoptosis ability of HeLa cells increased by the exogenous Tβ4 was attributed to an elevation of ROS.(30) However, little is known about the anti-oxidative role of endogenous Tβ4 in other human cancer cells. Hence, we addressed this question using human colon cancer cells as a model.

After Tβ4 downregulation in SW480 human colon cancer cells, increased superoxide anion was only slightly reduced by vitamin C treatment (Fig. S1), which was in partial agreement with an earlier report that the oxidized vitamin C transported into mitochondria by GLUT 10 can protect cells against oxidative stress.(31) In contrast, the elevation of ROS in SW480 cells triggered by Tβ4 knockdown was not inhibited by the treatment of several other commonly used antioxidants, suggesting that sustained oxidative stress is induced by Tβ4 knockdown in these cells. The above findings clearly indicate that neither endogenous anti-oxidative enzymes nor exogenous antioxidants (except vitamin C) could reduce significant increases of ROS in Tβ4-downregulating SW480 cells.

As the involvement of NOX was ruled out and an increase of superoxide anion was also detected in mitochondria (Fig. 2c), we postulated that these organelles are the major sources of ROS in Tβ4-downregulating SW480 cells. Supporting the aforementioned speculation, we found the presence of some abnormal mitochondria (either electron dense or cristae lost) in SW480 cells after Tβ4 knockdown (Fig. 2d). Furthermore, severe losses of mitochondrial membrane potential (Δψm) were also found in these cells (Fig. 3a,b), suggesting that certain mitochondrial functions were markedly diminished by Tβ4 downregulation in SW480 cells. In contrast, a modest but nonetheless significant increase of ATP and an elevated aerobic glycolysis, which leads to a higher level of lactate, were induced by Tβ4 knockdown in these cells (Fig. 4a,b). Intriguingly, both ATP and lactate levels in Tβ4-downregulating SW480 cells were back to normal in the presence of oxamate, an inhibitor of LDH (Fig. 4c,d), suggesting a compensatory increase of glycolyic capacity in human colon cancer cells after Tβ4 knockdown. In contrast to the notion that mitochondria-generated ROS function as a regulatory signal allowing adjustment of the cellular OXPHOS capacity,(19) as well as the concept that increased intracellular ROS concentrations are associated with increased mtDNA copy numbers,(21,32) we found a marked reduction of the subunits in OXPHOS complexes (except that of complex III) and dramatic decreases of mitochondrial DNA (mtDNA) in Tβ4-downregulating SW480 cells (Fig. 3). The latter might be accounted by the mutant p53 expressed in SW480 cells because a reduction of mtDNA resulting from the downregulation of p53R2, a subunit of ribonucleotide reductase, due to a loss of p53 was recently reported.(33) Collectively, although the precise mechanisms for the above-mentioned mitochondrial changes in Tβ4-knockdowned SW480 cells are not clear, excessive ROS produced by the defective mitochondria that cause oxidative damages to the mitochondrial constituents is speculated.(20)

In healthy cells, mitochondria continually divide and fuse to form a dynamic interconnecting network and the molecular machinery that mediates mitochondria fission and fusion is necessary to maintain its integrity, perhaps by facilitating DNA or protein quality control.(34,35) During apoptosis, cytochrome c release was found to be accompanied by mitochondrial fission and inhibition of Drp1-mediated mitochondrial fission reduced cytochrome c release. Even though the above findings suggest that inhibition of mitochondrial fission prevents the progression of apoptosis, this event alone does not necessarily result in cell death.(36) Moreover, disruption of F-actin attenuated fission and recruitment of Drp1 to mitochondria.(37,38) To our surprise, elevated fission of mitochondria was also detected in SW480 cells after Tβ4 knockdown (Fig. 5b), which might be attributed to a reduction in mtDNA even though F-actin disruption was found in these cells (data not shown). In addition to causing mitochondrial dysfunction and increasing apoptosis, Tβ4 downregulation also reduced the migration ability of Tβ4-overexpressing SW480 cells (Tb3 cells) (Fig. S7).(39) These findings strongly suggest a correlation between Tβ4 levels and mitochondrial integrity or invasion of colon cancer cells.

Our immunofluorescent staining results indicated the presence of Tβ4 in the mitochondria of SW480 cells. More interestingly, strong colocalization between Tβ4 and DsRed was also detected in H1299 and HCT116 cells (Fig. S8a), suggesting that Tβ4 is mainly localized in the matrix of mitochondria of both colon and lung cancer cells. The possible presence of Tβ4 in mitochondrion might be strengthened by a recent report that showed an interaction between Tβ4 and F1-F0 ATP synthase on the surface of endothelial cells.(40) In addition, increased superoxide anion and reduced mitochondrial membrane potential triggered by Tβ4 downregulation were also observed in HCT116 cells (Fig. S8b), suggesting that mitochondrial dysfunction might be a common event occurring in Tβ4-knockdowned human colon cancer cells.

Taken together, our data show, for the first time, that Tβ4 knockdown markedly increases intracellular ROS concurrently with a notable decline of mitochondrial function and a compensatory glycolysis. Even though a possible presence of Tβ4 in mitochondria is demonstrated, more work is needed to distinguish between a direct and an indirect role of this G-actin binding peptide in maintaining the integrity of mitochondria. Experiments of this nature are currently underway in our laboratory.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by grants NSC95-2320-B-010-043-MY3 and NSC98-2320-B-010-003-MY3, VGHUST95-P7-26 and VGHUST97-P6-21, and V97ER2-017 from the National Science Council of Taiwan, the Veterans General Hospital and University System of Taiwan, and the Taipei Veterans General Hospital, respectively, and a grant from the Ministry of Education, Aim for the Top University Plan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

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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