The HEK293, human colon cancer HT-29 and SNU-C4, and mouse colon cancer CT-26 cells were obtained from the Korean Cell Line Bank (Seoul National University, Seoul, Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, CA, USA). Cultures were maintained in a humidified atmosphere of 95% air/5% CO₂ at 37°C.

2′, 7′-dichlorofluorescein diacetate (DCFDA) and carboxyfluorescein succinimidyl ester (CFSE) were obtained from Molecular Probes (Carlsbad, CA, USA). The Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (San Jose, CA, USA). Anti-PGC-1α (sc-13067), anti-ACBP (sc-30190), anti-superoxide dismutase (SOD)-2 (sc-30080), anti-specificity protein 1 (Sp1) (sc-59), anti-rabbit IgG-FITC (sc-2012), anti-rabbit IgG-PE (sc-3739), and anti-mouse IgG-FITC (sc-2010) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-catalase antibody (ab1877) was purchased from Abcam (Cambridge, UK). The anti-FLAG^® (F7425), anti-β-actin (A1978), anti-rabbit IgG (A0545), and anti-mouse IgG secondary antibodies (A9044) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless stated otherwise, all other chemicals were purchased from Sigma.

HEK293 and CT-26 cells (1×10⁶) were transfected with 4 μg of pcDNA3.1-FLAG-PGC-1α expression vector from Spiegelman or empty vector (pcDNA 3.1) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedure. After transfection, stable cell lines were established after G418 selection (800 μg/ml) for 14 days. Stable cell lines were cultured in DMEM supplemented with 10% FBS, 800 μg/ml G418, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). Cultures were maintained in a humidified atmosphere of 95% air/5% CO₂ at 37°C.

Total RNA was extracted using RNAeasy mini kits (Qiagen, Valencia, CA, USA). Reverse transcription was performed using the RevertAid First Strand cDNA synthesis kit with a random hexamer as the primer (Thermo Scientific, Chicago, IL, USA). Real-time reverse transcriptase PCR (RT-PCR) was carried out on total RNA from HEK293 and HEK293-PGC-1α #1 cells transfected with PGC-1α and ACBP-specific or non-specific control small-interfering (si) RNA after 48 h on an ABI PRISM 7500 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA). Each real-time RT-PCR was done in triplicate according to the manufacturer’s instructions. TaqMan™ Gene expression assays [diazepam binding inhibitor (DBI)/ACBP (Hs01554584_m1)] were from Applied Biosystems. Quantification was based on a comparative ΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs03929097_g1) as endogenous control.

Cells were harvested, washed with phosphate-buffered saline (PBS), and treated with lysis buffer containing 20 mmol/l Tris (pH 8.0), 137 mmol/l NaCl, 10% glycerol, 1% Nonidet P-40, 10 mmol/l EDTA, 100 mmol/l NaF, 1 mmol/l phenylmethylsulfonyl fluoride and 10 mg/ml leupeptin. The lysates were centrifuged at 13,000 rpm for 15 min and the concentration of protein in each lysate determined using Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA, USA) following the manufacturer’s suggested procedure. Then, 8, 10 or 12% SDS-PAGE was used to separate 30 μg protein samples. Following electrophoresis, proteins were transferred to nitrocellulose membranes (Amersham Life Science, Inc., Arlington Heights, IL, USA). Blots were blocked overnight in 5% skim milk in PBS at 4°C and subsequently probed with primary antibody overnight. The blots were also probed with a monoclonal anti-β-actin antibody (Sigma) to be quantified as a relative loading control. Detection of specific proteins was carried out with enhanced chemiluminescence detection reagents (Amersham Life Science, Inc.) following the manufacturer’s instructions. Bands were quantified using Image Studio Lite Ver 3.1. (LI-COR, Inc., Lincoln, NE, USA)

HEK293, PGC-1α-HEK293, CT-26, and PGC-1α-CT-26 cells were seeded at a density of 1×10⁵/well in a 6-well plate. PGC-1α-HEK293 and SNU-C4 cells transfected with siRNAs for PGC-1α, ACBP, Sp1, or non-specific control siRNA were seeded at a density of 1×10⁵/well in a 6-well plate. After a 24-, 48- or 72-h culture, the cells were harvested by trypsinization using trypsin/EDTA and stained with trypan blue. The vital cells (those not stained with trypan blue) were counted under the microscope (Nikon Eclipse TS100; Nikon, Tokyo, Japan). Three independent experiments were carried out.

Cell proliferation was measured using the CFSE labeling assay as previously described (22). CFSE, which is used to fluorescently label live cells, is equally partitioned to daughter cells during division and can be used to measure cell proliferation. Briefly, cells were washed three times with PBS and incubated with 1 μM CFSE dye (Molecular Probes) for 15 min. The cells were then washed again, incubated with fresh medium containing 10% FBS, and seeded in 6-well plates at a density of 1×10⁵ cells/well. Cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences) after culture for 24, 48 or 72 h. Each condition was performed in triplicate.

Total RNA extracted from HEK293 cells and stable HEK293 cells expressing PGC-1α was used for the synthesis of first-strand cDNA by reverse transcriptase. Reverse transcription was performed for 1.5 h at 42°C in a final reaction volume of 20 μl containing 3 μg of the purified total RNA, 4 μl of 5X reaction buffer (Promega, Madison, WI, USA), 5 μl of dNTPs (each 2 mM), 2 μl of 10 μM dT-ACP1(5′-CTGTGAATGCTGCGACTACGATIIIIIT(18)-3′), 0.5 μl of RNasin RNase Inhibitor (40 U/μl; Promega), and 1 μl of Moloney murine leukemia virus reverse transcriptase (200 U/μl; Promega). First-strand cDNAs were diluted by the addition of 80 μl of ultra-purified water for the GeneFishing™ PCR and stored at −20°C until use.

Differentially expressed genes were screened using the ACP-based PCR method (23) with the GeneFishing DEG kits (Seegene). Briefly, second-strand cDNA synthesis was conducted at 50°C during one cycle of first-stage PCR in a final reaction volume of 20 μl containing 3–5 μl (~50 ng) of diluted first-strand cDNA, 1 μl of dT-ACP2 (10 μM), 1 μl of 10 μM arbitrary ACP and 10 μl of 2X Master Mix (Seegene). The PCR protocol for second-strand synthesis was one cycle at 94°C for 1 min, followed by 50°C for 3 min and 72°C for 1 min. After second-strand DNA synthesis was completed, the second-stage PCR amplification protocol was 40 cycles of 94°C for 40 sec, followed by 65°C for 40 sec and 72°C for 40 sec and a 5-min final extension at 72°C. The amplified PCR products were separated on a 2% agarose gel stained with ethidium bromide. The amplified cDNA fragments with >2-fold differential band intensities were re-amplified and extracted from the gel by using the GeneClean II kit (Qbiogene, Solon, OH, USA), and directly sequenced with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

The extent of apoptosis was evaluated by Annexin V-FITC and flow cytometry as previously described (24). The Annexin V assay was used, in which Annexin was conjugated with FITC. Propidium iodide (PI) was used as counterstain. Briefly, cells were treated with 0.5, 1 or 2 mM H₂O₂ for 24 h. After incubation, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with Annexin V-FITC (BD Pharmingen, San Diego, CA, USA) and 2 μg/ml PI in binding buffer (10 mM HEPES, pH 7.4/140 mM NaCl/2.5 mM CaCl₂) for 15 min at 37°C in the dark. The samples were analyzed by flow cytometry using a FACScan flow cytometer. Data analysis was performed using CellQuest software (Becton-Dickinson, San Jose, CA, USA).

HEK293 cells (2.0×10⁴) overexpressing PGC-1α or empty vector were suspended in 1 ml of 0.3% agarose (Sigma) and then added to 1- of a 6-well plate with a foundation layer of 0.5% agarose in triplicate. Approximately 24 h later, the cells received an additional 1 ml of growth medium before incubation for a further 14 days. Colonies were stained with 0.01% crystal violet and counted under the microscope (Nikon) in 20 fields of ×10 magnification. Light microscopy images were captured under ×100 magnification.

All animal procedures and care were approved by the Institutional Animal Care and Usage Committee of Dong-A University. To determine the effect of PGC-1α on tumor formation, viable HEK293 cells (2×10⁷ cells/100 μl) overexpressing PGC-1α or empty vector were subcutaneously injected into the bilateral flanks of 6- to 7-week-old female nu/nu immunodeficient BALB/c mice, respectively (Orient Bio Inc., Seongnam, Korea). Tumor size was measured daily with a caliper (calculated volume = shortest diameter² × longest diameter/2). The mice were followed for tumor size and sacrificed on the 45th day. Tumors were resected, weighed, and frozen or fixed in formalin and paraffin-embedded for immunofluorescence staining.

Cells were cultured on a Lab-Tek^® Chamber Slide™ (Nalgen Nunc Inc., Rochester, NY, USA) and then fixed with 3% formaldehyde and permeabilized using 0.01% Triton X-100. After being blocked with 3% FBS for 30 min, cells were incubated in primary antibody for 1 h followed by a fluorescence-labeled secondary antibody (Sigma) for 30 min. Cells were then washed, mounted using glycerol, and analyzed by confocal microscope (Zeiss LSM 510 confocal microscope; Carl Zeiss Co., Ltd., Jena, Germany) using a 40× C-Apochromat objective. Negative control staining was performed with secondary antibodies alone.

ROS production was monitored by flow cytometry using carboxy-H₂DCFDA (Molecular Probes). PGC-1α-HEK293 and SNU-C4 cells transfected with siRNAs for PGC-1α, ACBP, or Sp1, or with non-specific control siRNA were washed twice with PBS to remove the extracellular compounds. H₂DCFDA (100 μmol/l) was added for an additional hour. Green fluorescence was excited using an argon laser and detected using a 525-nm band-pass filter by flow cytometric analysis.

The siRNA sequence used for the targeted silencing of PGC-1α, ACBP, or Sp1 was designed by Qiagen (GS10891), Dhamacon(L-006488-00-0005, ThermoScientific, Chicago, IL, USA), or Santa Cruz (sc-29487), respectively. For transfection, cells were resuspended at 1.3×10⁷/0.5 ml in PBS and mixed with 4 nM siRNA for PGC-1α, 4 nM siRNA for ACBP, or 4 nM siRNA for Sp1, or with 4 nM non-silencing siRNA using Lipofectamine 2000 (Invitrogen) following the manufacturer’s procedure. After transfection, cells were cultured in 10% FBS-supplemented DMEM for 48 h. These cells were then used for cell proliferation, Annexin V-PI staining, assessment of ROS production, immunofluorescence staining, real-time RT-PCR and western blot analysis.

PGC-1α-HEK293 #1 cells were washed in PBS and centrifuged at 1,000 rpm for 5 min to harvest the cells. Ice-cold modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na₃VO₄, 1 mM NaF) was added to the cells, which were gently rocked on a rocker at 4°C for 15 min for cell lysis. The lysate was centrifuged at 14,000 rpm for 15 min at 4°C and pre-cleared by addition of 100 μl of 50% Protein G sepharose bead slurry per 1 ml of cell lysate. The supernatant was incubated with specific antibody overnight at 4°C with gentle rocking. The immune complex was captured by the addition of 100 μl Protein G sepharose (Invitrogen). The sepharose beads were washed three times with 800 μl ice-cold RIPA buffer and resuspended in 60 μl 2X sample buffer, followed by elution through boiling. The immune complexes were fractionated on 10% SDS-PAGE followed by western blotting with appropriate antibodies.

Statistical analyses were done with the SPSS 21.0 statistical package for Windows (SPSS, Chicago, IL, USA). Data are expressed as mean values ± standard deviation (SD). One-way ANOVA was used to determine whether there were significant differences in cell viability between PGC-1α-HEK293 #1 cells, PGC-1α-HEK293 #3 cells and HEK293 wild-type cells. Differences in tumor volumes between control HEK293 cell-injected and PGC-1α-HEK293 cell-injected groups were evaluated using the unpaired Student’s t-test. Statistical significance was defined as P<0.05.

As a first step toward assessing the functional significance of PGC-1α overexpression for growth of cells, HEK293 and CT-26 cells were transiently transfected with a FLAG-tagged PGC-1α expression vector or with the parental vector (pcDNA 3.1) and cultured with G418 (800 μg/ml) for 14 days. After that, several colonies were picked and amplified. Exogenously expressed PGC-1α was detected by western blotting and immunofluorescence staining with antibodies against PGC-1α and FLAG. As shown in Fig. 1A, the protein of PGC-1α in PGC-1α-HEK293 #1, PGC-1α-HEK293 #3, and PGC-1α-CT-26 #9 cells was increased compared to wild-type or pcDNA-transfected HEK293 and CT-26 cells. In addition, the exogenously expressed FLAG-tagged PGC-1α (FLAG-PGC-1α) was detected in PGC-1α-transfected HEK293 (PGC-1α-HEK293 #1 and #3) and PGC-1α-transfected CT-26 (PGC-1α-CT-26 #9) cells (Fig. 1B).

To investigate the effect of PGC-1α overexpression on growth of HEK293 and CT-26 cells, cell viability and proliferation were determined by cell counting and CFSE labeling assays. As shown in Fig. 1C, the average number of cells in PGC-1α-transfected HEK293 and CT-26 cells (PGC-1α-HEK293 #1 and #3; 16.1×10⁵ and 15.5×10⁵ at 72 h, respectively; PGC-1α-CT-26 #9; 11.9×10⁵ at 72 h) was significantly higher than in wild-type HEK293 and CT-26 cells (3.9×10⁵ at 72 h; 7.9×10⁵ at 72 h, respectively). In addition, as expected, the number of cells with high fluorescence decreased with days in culture, so that the peak signals for PGC-1α-transfected cells (PGC-1α-HEK293 #1, #3 and PGC-1α-CT-26 cells) were shifted to the left compared to control cells (Fig. 1D). Thus, CFSE labeling analysis confirmed that PGC-1α overexpression enhances cell proliferation of HEK293 and CT-26 cells.

The effect of PGC-1α on cell proliferation was further investigated using siRNA transfection to knock down endogenous PGC-1α expression in human colorectal cancer HT-29 and SNU-C4 cells. At 72 h after transfection, PGC-1α expression was 40 and 53% lower than in wild-type or non-specific control siRNA-transfected HT-29 and SNU-C4 cells, respectively (Fig. 2A and B). Concurrently, proliferation of PGC-1α siRNA-transfected HT-29 and SNU-C4 cells at 48 h post-transfection was significantly suppressed in comparison with that of untreated or non-silencing siRNA-transfected cells (37.2 and 33.5% reduction, respectively; P<0.01) by cell counting and CFSE labeling (Fig. 2C). In addition, PGC-1α siRNA transfection repressed cell proliferation by 26% at 48 h post-transfection in PGC-1α-HEK293 #1 cells (Fig. 2C). Taken together, these results support an important role of PGC-1α in the regulation of cell proliferation.

Because anchorage-independent growth is considered to be an in vitro test for tumorigenesis, we assayed the effects of overexpression of PGC-1α on the ability of HEK293 cells to form colonies in soft agar. As shown in Fig. 3A, overexpression of PGC-1α in HEK293 cells resulted in a higher incidence of colony formation than observed following transfection with the empty vector (114. 4±13.6 and 67.8±6.6, P<0.001, respectively). The cells transfected with empty vector formed no colonies during 14 days of culture whereas both cell lines expressing PGC-1α formed a significantly high number of colonies of a large size (Fig. 3A). In addition, to confirm the impact of PGC-1α overexpression on tumorigenesis, wild-type HEK293 cells or PGC-1α-overexpressing HEK293 cells were injected subcutaneously into bilateral flanks of immunodeficient Balb/c mice, and tumor development and size were examined. Wild-type HEK293 cells formed tumors at a low percentage (6.67±6.67%) whereas the matched PGC-1α-HEK293 cells (PGC-1α-HEK293 #1 and PGC-1α-HEK293 #3) formed tumors in 76.67±8.82 and 60.00±15.30% of mice, respectively (Fig. 3B and C). In addition, the average tumor size in mice with PGC-1α-HEK293 #1 (652.1±263.5 mm³) was larger than in mice with PGC-1α-HEK293 #3 (217.3±75.9 mm³), suggesting that the expression level of PGC-1α is related to tumor growth. Furthermore, the presence of tumor cells was confirmed by hematoxylin and eosin staining (Fig. 3D). Thus, these data suggest that PGC-1α overexpression dramatically enhances the tumorigenic capacity of HEK293 cells in Balb/c nude mice.

Previous studies have demonstrated that PGC-1α expression leads to expression of antioxidants such as SOD and catalase (10,26) and provided evidence that PGC-1α can protect neuroblastoma cells from H₂O₂-mediated cell death (10). In this study, to evaluate the role of PGC-1α in providing a defense against oxidative stress, wild-type HEK293 and PGC-1α-overexpressing HEK293 cells were treated with various concentrations of H₂O₂ for 24 h; the extent of apoptosis was determined using Annexin V-PI staining. Approximately 72.7% of the empty vector-expressing HEK293 cells died after exposure to 1 mM H₂O₂, but only 28.7 and 29.4% of the PGC-1α-transfected HEK293 cells (PGC-1α-HEK293 #1 and PGC-1α-HEK293 #3) died, respectively (Fig. 4A). Consistent with these changes, wild-type HEK293 cells had significantly higher intracellular levels of ROS than PGC-1α-transfected HEK293 cells did (5.64±0.55-vs. 1.91±0.22- and 2.38±0.30-fold, P<0.01 and P<0.05, respectively), as indicated by increased fluorescence in the presence of the compound DCF-DA (Fig. 4B).

In addition, the basal level of ROS was determined in wild-type, PGC-1α-HEK293 #1 and PGC-1α-HEK293 #3 cells. The basal level of ROS was reduced in PGC-1α-transfected cells compared with wild-type HEK293 cells (0.60±0.03- and 0.57±0.01-fold vs. 1, respectively) (Fig. 4B). The reduced basal ROS of PGC-1α-HEK293 cells may be related to the increased expression of antioxidants. The expression of catalase was significantly 2.62- and 2.52-fold increased in PGC-1α-transfected HEK293 (PGC-1α-HEK293 #1 and #3) cells, respectively (Fig. 4C). The expression of SOD was significantly 1.85- and 1.62-fold increased in PGC-1α-transfected HEK293 (PGC-1α-HEK293 #1 and #3) cells, respectively (Fig. 4C). To confirm these results, the extent of H₂O₂-induced apoptosis and ROS level were measured in PGC-1α-siRNA- or non-specific control siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells. The extent of H₂O₂-induced apoptosis and ROS level were significantly increased in PGC-1α-siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 4D and E). The increased basal ROS of PGC-1α-siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells may be caused by the decreased expression of antioxidants. Expression of SOD and catalase was decreased in PGC-1α-siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 6A). These results indicate that PGC-1α can protect HEK293 cells from oxidative stress such as that caused by H₂O₂. Decreased susceptibility to ROS-induced apoptosis may contribute to increased cell proliferation in PGC-1α-HEK293 cells.

To investigate genes that contribute to PGC-1α-induced cell proliferation and tumorigenesis, ACP-based GeneFishing PCR was performed, and the band intensities between PGC-1α-transfected HEK293 cells and empty vector-expressing HEK293 cells were compared. By densitometric analysis of amplified cDNA fragments, one fragment showing >2-fold different intensities between two cell lines was observed (Fig. 5A). The amplicon band was eluted from agarose gel, re-amplified, and sequenced, and the sequences were used in a BLAST search to identify their gene annotations. BLAST analysis identified the ACBP.

To confirm the upregulated expression of ACBP in PGC-1α-overexpressing HEK293 cells, real-time RT-PCR, immunoblot analysis, and immunofluorescence staining were performed. As shown in Fig. 5B and C, mRNA and protein levels of ACBP were approximately 2.4- and 2-fold (mRNA) and 2.09- and 1.99-fold (protein) higher in PGC-1α-HEK293 #1 and #3 cells, respectively, than in wild-type HEK293 cells. In addition, immunofluorescence staining showed that expression levels of ACBP were increased in PGC-1α-overexpressing HEK293 cells and that most of the ACBP was co-localized with PGC-1α (Fig. 5D). Furthermore, the expression of ACBP was decreased in PGC-1α siRNA-transfected PGC-1α-HEK293 #1 cells based on western blot results (Fig. 6A). However, the expression of PGC-1α was not affected by ACBP siRNA knockdown (Fig. 6A). Thus, these data indicate that the expression of ACBP may be regulated by the activity of PGC-1α.

The above-described findings indicated that ACBP expression was regulated by PGC-1α. To examine the involvement of ACBP in regulating cell proliferation by PGC-1α expression, ACBP was downregulated in an ACBP siRNA knockdown experiment in PGC-1α-transfected HEK293 cells and SNU-C4 cells. In PGC-1α-transfected HEK293 and SNU-C4 cells, the endogenous expression of ACBP was substantially reduced following transfection with siRNA for ACBP (Fig. 6A); whereas, the proliferation of ACBP siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells were significantly inhibited compared to control PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 6B; 44 and 30% reductions, respectively). However, protein levels of PGC-1α were not downregulated in ACBP siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 6A).

In addition, to evaluate the role of ACBP in the regulation of sensitivity to H₂O₂-induced apoptosis, the extent of H₂O₂-induced apoptosis and ROS level were measured in ACBP siRNA-transfected or non-silencing siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells. The extent of H₂O₂-induced apoptosis and ROS level were significantly increased in ACBP siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells compared to control PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 6C). Expression of SOD and catalase was decreased by ACBP siRNA knockdown in PGC-1α-HEK293 and SNU-C4 cells (Fig. 6A). These results indicate that ACBP can protect PGC-1α-HEK293 #1 and SNU-C4 cells from oxidative stress such as that mediated by H₂O₂. Increased susceptibility to ROS-induced apoptosis may contribute to decreased cell proliferation by ACBP siRNA knockdown in PGC-1α-HEK293 #1 and SNU-C4 cells. The decreased cell proliferation and increased sensitivity to H₂O₂-induced apoptosis by ACBP knockdown is similar to that observed in PGC-1α siRNA-transfected PGC-1α-HEK293 #1 and SNU-C4 cells. Thus, these results suggest that PGC-1α knockdown downregulates cell proliferation and increases H₂O₂-induced apoptosis of HEK293 and SNU-C4 cells through downregulation of ACBP.

The above results suggested that PGC-1α expression influences ACBP expression, but interactions between PGC-1α and ACBP have not previously been described. Thus, we tested the possibility that PGC-1α can interact with ACBP. Using antibodies against FLAG, we found that FLAG antibody did not immunoprecipitate ACBP (Fig. 7A). Consistent with this outcome, ACBP antibodies did not immunoprecipitate FLAG-PGC-1α (Fig. 7B). These results indicate that PGC-1α did not directly interact with ACBP.

The results described above showed that ACBP is a target of PGC-1α; however, the mechanisms for its regulation by PGC-1α are not clear. A previous report suggests that the promoter of ACBP has an Sp1 binding site (27). Thus, we investigated whether PGC-1α expression can influence the expression of Sp1. The results indicated that the expression of Sp1 was significantly 3-fold increased in PGC-1α-HEK293 #1 and #3 cells compared to wild-type HEK293 cells (Fig. 8A). In addition, PGC-1α siRNA transfection led to reduced expression of Sp1 in PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 6A).

The knockdown of Sp1 in PGC-1α-HEK293 #1 and SNU-C4 was confirmed by western blot analysis. The expression of Sp1 was decreased in PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 8B). Knockdown of Sp1 resulted in decreased cell proliferation, decreased expression of ACBP, decreased expression of catalase and SOD, increased ROS production, and increased H₂O₂-induced apoptosis in PGC-1α-HEK293 #1 and SNU-C4 cells (Fig. 8B–D), which is similar to those observed in PGC-1α siRNA-transfected PGC-1α-HEK293 #1 cells. Taken together, these data suggest that the increased ACBP expression by PGC-1α may be caused by increased Sp1 expression.