B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NFκB pathways which enhances transcription of MnSOD by accelerating IκBα degradation in normal and cancer cells

Background B-cell translocation gene 2 (BTG2) belongs to antiproliferative (ARPO) gene family and the expression of BTG2, human ortholog of rat PC3 and mouse TIS21 gene, has been shown to render cancer cells more sensitive to doxorubicin treatment by upregulating MnSOD expression without regulating any other reactive oxygen species (ROS) scavenging enzymes. Results In the present study, by employing exogenous and endogenous BTG2/TIS21/Pc3 expression by transfection and transduction analyses, and by knockdown of gene expression using RNA interference or using gene knockout cells, we observed that BTG2 increased the binding of activated NF-κB (p65/RelA) to the enhancer element of MnSOD gene in the 2nd intron, which was regulated by p-Akt1, and the induction of MnSOD by BTG2 was accompanied with subsequent downregulation of ROS level and cyclin B1 biosynthesis along with the increase of p21WAF1, resulting in the G2/M arrest independent of p53. Conclusions These results show for the first time that BTG2 mediates crosstalk between PI3K-Akt1 and NF-κB pathways, which regulates p53-independent induction of G2/M phase arrest both in normal and cancer cells.


Background
Human BTG2 belongs to the antiproliferative (APRO) gene family along with its orthologs, rat PC3 and mouse TIS21 [1]. It potentiates NGF-induced differentiation of PC12 cells and protects neurons from apoptosis [2]. In contrast, overexpression of BTG2 significantly enhances reactive oxygen species (ROS) generation after doxorubicin treatment of HeLa cells via burst of H 2 O 2 by induced manganese-containing superoxide dismutase (MnSOD) without regulating catalase and glutathione peroxidase expression [3]. The APRO gene, including TIS21 [4], PC3 [5] and BTG2 [6], has originally been reported as a primary response gene transiently expressed in the established cell lines in response to various stimulations, however, BTG2 /TIS21/PC3 (BTG2) expression in mouse and human tissues has been shown to be rather constitutive [7], and it is found to be significantly reduced during carcinogenesis in the epithelial cells of thymus [8], prostate [9], kidney [10], breast [11], liver [12] and brain [13]. Furthermore, the loss of BTG2 expression is related significantly with tumor grade, metastasis and resistance to cancer treatment, especially in the estrogen receptor positive breast cancers [14]. In addition, BTG2 regulates cancer cell migration via regulations of ROS level and chemokines Cxcl3 and Cxcl12 [13,15]. It has been reported that BTG2 plays a major role at the downstream of p53 gene [16], inhibits Ras-induced cell transformation [17] by inhibiting its activation to RasGTP via direct interaction with Ras protein [18]. Therefore, BTG2 is also involved in the regulation of mutant p53 activities via inhibiting Ras-related cancer gene signatures [19]. All the above mentioned findings together strongly suggest the important roles of BTG2 played in carcinogenesis. BTG2 also regulates both G1/S and G2/ M phase arrest independent of p53 and pRB expression; G1/S phase arrest is regulated by inhibiting synthesis of cyclin E and cdk4 activity independent of pRB [20], whereas BTG2 regulates G1/S progression in pRB dependent manner by reducing cyclin D1 expression [21]. Furthermore, BTG2 strongly induces G2/M phase arrest in U937 myelomonocytic leukemia (p53 null) and Huh7 (p53 mutant) hepatoma cells [22], therefore, we earlier suggested BTG2 as a pan-cell cycle inhibitor independent of p53 and pRB activities via interaction with Pin1 in response to EGF stimulation [23].
Several reports suggest that BTG2 protein is a general activator of mRNA deadenylation. BTG2 overexpression caused accelerated deadenylation of reporters and of endogenous transcripts. It directly interacts with Caf1 which is a component of the CCR4-NOT deadenylase complex. These observations for the first time showed the role of BTG2 in the general control of mRNA decay [24][25][26]. As mentioned above, BTG2 enhances MnSOD expression in HeLa cells after treatment with doxorubicin [3] and MnSOD has been suggested as a new type of tumor suppressor [27]. Overexpression of MnSOD inhibits the growth of a wide variety of cancer cells and protects oxygen-utilizing cells from the toxicity of ROS under physiologic condition [28]. Furthermore, MnSOD expression is regulated at the transcriptional, translational and posttranslational levels, and Sp1 and AP-2 are reported to be homeotic transcriptional regulators of MnSOD [29], whereas induction of MnSOD expression requires the binding of NF-κB transcription factor (p65) to the enhancer element in the intron-2 of MnSOD gene [30]. In addition, acetylation of MnSOD influences its enzymatic activity in response to nutrient status or oxidative stress at the posttranslational level [31].
In the present study, we attempted to answer the questions of whether MnSOD can be a direct transcriptional target of BTG2 or not, what is the mechanism of MnSOD induction by BTG2, what kinds of signal pathways regulate the induction of MnSOD expression, and what is outcome of MnSOD induction by BTG2 in cancer and normal cells. In fact, various lines of evidences suggest that free radical-scavenging agents inhibit neoplastic processes at the cellular and molecular levels [32]. Tumor cells are low in MnSOD activity and its expression, compared to normal counterparts [27], therefore, tumor cells may particularly be able to accumulate higher levels of superoxide or hydrogen peroxide than the normal, which may enhance survival and proliferation of the cancer cells. We observed in the present study that the expression of MnSOD was upregulated by BTG2 at the transcription level via degradation of IκBα in cancer and normal cells, and that underlying mechanism of APRO effect of BTG2 involved the crosstalk between PI3K-Akt1 and NFκB pathways, which resulted in the scavenge of ROS and increased p21 WAF1 expression.

BTG2 upregulates MnSOD expression in HeLa cells
We earlier showed that BTG2 sensitizes HeLa cells to doxorubicin treatment via induction of endogenous MnSOD expression [3]. To investigate molecular mechanism involved in MnSOD induction by BTG2, we overexpressed BTG2 gene in HeLa cells by DNA transfection and viral transduction, and found that BTG2 upregulated MnSOD expression in the dose-dependent manner both in its mRNA ( Figure 1A and 1C) and protein ( Figure 1B) levels. On the other hand, endogenous level of MnSOD expression in the TIS21 −/− MEF along with wt-MEF was much lower in the TIS21 −/− MEF than the control ( Figure 1D), suggesting that the effect of BTG2 on MnSOD expression was both endogenously and exogenously regulated in mouse fibroblasts and cancer cells, and that there is a possibility of MnSOD gene as a direct target of BTG2.

BTG2 activates NFκB-response element of MnSOD gene in the 2nd intron
To investigate a mechanism of MnSOD induction by BTG2 in HeLa cells, we performed luciferase assay using the promoter DNA construct (−2215 to +42) of MnSOD gene ligated to the upstream of luciferase, however, there was no difference in the promoter activity by the expression of BTG2 gene (Figure 2A), except by Sp1, the positive control. Since inducible expression of MnSOD has been shown to be regulated by NFκB binding to the enhancer element of MnSOD gene [30], we subcloned the NF-κB response element (κB-RE) into the pGL3-BASIC vector and performed luciferase analysis with or without BTG2 coexpression. As shown in Figure 2B, the luciferase activity was increased with BTG2 coexpression, suggesting that BTG2 enhances the activity of κB-RE in the MnSOD gene. Proteins of the transfected genes were monitored by immunoblot analyses and presented under the Figure 2A and 2B. To rule out other possibility of MnSOD induction by BTG2 expression, the activity of FOXO3a, a well known transcription factor of MnSOD in quiescent cells against oxidative stress [33], was analyzed by measuring its phosphorylation on serine253 residue, however, there was no significant difference of its activity after BTG2 expression (Additional file 1: Figure S1A). To further investigate the BTG2-mediated activation of κB-RE of MnSOD gene, the activation of NFκB pathway in HeLa cells was evaluated by measuring the degradation of IκBα in proteasome [34]. Increasing amounts of BTG2 expression significantly enhanced the degradation of IκBα protein along with MnSOD expression ( Figure 2C). Moreover, when the cells were treated with MG132 to block ubiquitin proteasome pathway, BTG2-mediated MnSOD induction was accordingly reduced ( Figure 2D). The data support our observation in Figure 2B that BTG2 increased transcription of MnSOD through the activation of κB-RE. To further confirm the activation of NFκB by BTG2, we performed ChIP analysis using the cell lysates obtained from the BTG2 and the LacZ expressers. As expected, the interaction of p65 with κB-RE was observed only in the BTG2 expresser, but not in the control ( Figure 2E, lower panel). Since TPA has been known to induce MnSOD through NFκB pathway [35,36], TPA treatment (100 ng/mL for 2 h) was employed as a positive control for the ChIP analysis. As expected, TPA increased IKBα degradation (Additional file 1: Figure S1B) and induced binding of p65 to κB-RE of MnSOD gene ( Figure 2E, upper panel). To further evaluate the upstream kinase of IκBα, phosphorylation of IKKα/β on serine176 residue was examined, and its minimal activation was observed along with significant degradation of IκBα in the BTG2 expresser without any other antioxidant scavenging enzymes such as catalase, GPX, or SOD1 ( Figure 2F).

BTG2 mediated-NFκB activation is regulated by IκBα degradation
To further confirm the specificity of BTG2 effect on MnSOD expression via NFκB activation, HeLa cells were transfected with short interfering RNAs against BTG2 (siBTG2), and the changes of MnSOD and IκBα expression were evaluated by RT-PCR and immunoblot analyses, respectively. Knockdown of endogenous BTG2 downregulated MnSOD level ( Figure 3A), whereas IκBα was further accumulated ( Figure 3B). Moreover, transfection of siBTG2 also reduced the effect of exogenous BTG2 on the degradation of IκBα protein in the cells ( Figure 3C). To confirm the effect of IκBα degradation on the BTG2regulated MnSOD induction, successive transductions of the lentivirus with nondegradable IκBα mutant and the Ad-BTG2-HA were performed. As shown in Figure 3D, the BTG2-induced MnSOD expression was reduced by coinfection with IκBα-mutant which was unable to be disrupted ( Figure 3E), supporting the activation of NFκB by BTG2 is specific and mediated via IκBα degradation.

BTG2-enhanced IκBα degradation is regulated by p-Akt1
We have earlier shown that the activation of Akt in response to estradiol requires BTG2 expression in MEF and bone marrow precursor cells (Lin --c-Kit + -Sca1 + ) [37]. Therefore, the possibility of crosstalk between PI3K-Akt and NFκB pathways was explored. When HeLa cells were transfected with BTG2, Akt phosphorylation on Serine473 was significantly increased ( Figure 4A). To further explore the crosstalk between pAkt and IκBα degradation, PI3K inhibitors (LY294002 and Wortmanin) were applied to the  Figure 4 Regulation of BTG2-enhanced IκBα degradation via activation of PI3K-Akt1. Based on our previous report [37], upstream signals of the IκBα degradation by BTG2 were investigated. (A) Immunoblot analysis showing the significant phosphorylation of Akt at Ser 473 residue in the BTG2 expresser. To explore whether there is a crosstalk between PI3K and NFkB pathways, HeLa cells transfected with BTG2 were treated with PI3K inhibitors, LY294002 (B) and Wortmanin (C), respectively, and then regulation of IκBα degradation was examined by immunoblot analyses. Note significant inhibition of BTG2-mediated IκBα degradation after treatment with PI3K inhibitors, suggesting the cross-talk between PI3K-Akt and NFκB pathways by the expression of BTG2. (D) RT-PCR revealing the specific knockdown of Akt1, not Akt2, by the short interfering RNAs to Akt1. (E) BTG2-mediated IκBα degradation was significantly inhibited by transfection of siAkt1, whereas transfection of siControl failed to change IκBα degradation at all, supporting the specific effect of Akt1 on the activation of BTG2-mediated NFκB pathway. (F) To further specify the effect of Akt1 on the BTG2-mediated IκBα degradation, transfection of siAkt1 RNA was combined with Ad-BTG2 infection in HeLa cells, and then ChIP analysis was performed with anti-p65 antibody. As expected, interaction of p65 with κB-RE was significantly reduced in the BTG2 and siAkt1 coexpressers (1.9 vs. LacZ and siControl) than that of the BTG2 alone expresser (3.3 vs. LacZ and siControl), indicating downregulation of NFκB activation by siAkt1 (over 40%) and the activity of Akt1 at the upstream of NFkB activation in the presence of BTG2 expression. Inhibition of p65 binding to κB-RE after transfection of siAkt1 was quantified by Image J software, and the relative densities of kB-RE found in the ChIP assay based on those of the Input (ChIP/Input) were showed below the Figure 4F. The experiment was repeated (n = 3).
test system and the effect of PI3K inactivation on the expression of IκBα and Akt activation was examined. The result in the 3 rd and 4 th lanes in Figure 4B and 4C clearly showed the regulation of NFκB activation by PI3K-Akt signal. To further specify the role of Akt1 in the activation of NFκB pathway, HeLa cells were transfected with siAkt1 ( Figure 4D) and the inhibition of BTG2-mediated IκBα degradation was examined by immunoblot analysis. As expected, knockdown of Akt1 recovered BTG2-mediated degradation of IκBα expression in the siAkt1 and BTG2 cotransfected cells ( Figure 4E). Moreover, ChIP assay revealed inhibition of p65 binding to κB-RE up to 40% by cotransfection of siAkt1 and BTG2 (3 rd and 4 th lanes in Figure 4F). These data strongly suggest the BTG2-mediated Akt1 activation at the upstream of IκBα degradation.
To exclude the possibility that the crosstalk is a phenomenon limited to HeLa cells, A549 human lung cancer cells were employed and confirmed the induction of MnSOD by transfection of BTG2 ( Figure 5A) with concomitant degradation of IκBα ( Figure 5B). Moreover, the specific binding of p65 to κB-RE was also observed in the BTG2 expressers by ChIP assay (Figure 5C). At the same time, the crosstalk between pAtk1 and NFκB was confirmed by employing knockdown of Akt1 in the same cells ( Figure 5D). Furthermore, BTG2 mediated-IκBα degradation was confirmed also in MCF7 breast cancer cells and wt-MEF after transfections of BTG2 cDNAs and siBTG2 /TIS21 , respectively (Additional file 2: Figure S2). All these data strongly suggest the regulation loop between BTG2-PI3K/Akt1-NFκB pathways for the induction of MnSOD expression.

BTG2 enhances G2/M arrest along with reduction of H 2 O 2 level
To explore physiologic significance of BTG2 overexpression, HeLa cells were synchronized at the late G1 and early S phases by thymidine double blocks and the cell cycle progression was monitored for 12 h by FACS analysis after the release. Figures 6A (X-axis corresponds to DNA content and Y-axis represents number of event) and 6C revealed that the expression of cyclin B1 was delayed along with G2/M arrest in the BTG2 expressers at 4 h after the release and the phenomenon is well accordant with our previous report observed in U937 cells [22]. When the percentage of cells in G1 and G2 phases were calculated using the ModFit software, Figure 6B     with the LacZ control.To further evaluate whether the G2/M arrest in the BTG2 expresser is restricted only to cancer cells or not, we analyzed cell cycle progression of NIH3T3 cells infected with adenovirus carrying either BTG2 or LacZ control and synchronized at G1/S phases by using thymidine double block (Additional file 3: Figure S3A). There was no significant differences of G2/M phase progression and cyclin B1 expression in the NIH3T3 cells (Additional file 3: Figures S3B-S3D), suggesting that induction of G2/M arrest by BTG2 overexpression might be limited to cancer cells which usually contain a defect in G1/S checkpoint, BTG2mediated MnSOD induction was preceded to p21 WAF1 expression ( Figure 6D) and the expression of p21 WAF1 was evident both in the RNA and protein levels ( Figure 6E and Additional file 4: Figure S4) after transduction and transfection of BTG2 gene in HeLa cells. Furthermore, BTG2 expresser scavenged intracellular H 2 O 2 level along with cell cycle progression from G1/S to G2/M phase ( Figure 6F). All the results can be supported by the notion that NFκB enhances G2/M arrest via induction of p21 WAF1 [38].

Discussion
In the present study, we presented evidences on the crosstalk between PI3K-Akt and NFκB pathways after ectopic and endogenous BTG2 expressions in normal and cancer cells via activation of Akt1 and degradation of IκBα protein. The signals regulating MnSOD expression -the binding of NFκB (p65) to the enhancer element of MnSOD gene in response to BTG2 expression -significantly reduced ROS level and cyclin B1 biosynthesis as opposed to p21 WAF1 induction, which resulted in G2/M arrest (Figure 7). It has been known that the homeotic expression of MnSOD is regulated by AP-2 and SP-1 binding to the promoter of MnSOD gene [29], whereas induced expression of MnSOD requires enhancer activation located in the 2 nd intron [30]. Indeed, the upregulation of BTG2-enhanced MnSOD expression might be one of the intracellular cooperation between APRO gene and a tumor suppressor through the regulations of cell division cycle and intracellular ROS level, since the notion was supported by luciferase assays using promoter and enhancer element of MnSOD gene and by reverse correlations of IκBα and MnSOD expression, in addition to ChIP and immunoblot analyses ( Figure 2). Specificity of the effects was further confirmed by RNA interference and IκBα (Ser32, Ser36 to Ala32, Ala36) mutant analysis in HeLa cells ( Figure 3) and A549 cells ( Figure 5) in addition to the wt-MEF and MCF 7 breast cancer cells (Additional file 2: Figure S2). Our present results are well supported by a recent study that BTG2 works as a coactivator of the antioxidant transcription factor, NFE2L2, which induces antioxidant gene expression including catalase and superoxide dismutases 1 and 2, thus BTG2 is able to protect human mammary epithelial cells from oxidative stress by H 2 O 2 and other oxidants [39]. In contrast to our present result, HeLa cells have been shown to enhance cell death by H 2 O 2 burst along with MnSOD induction when the cells are exposed to toxic dose (1.0 μg/ml) of doxorubicin [3]. Therefore, it is highly likely that the BTG2mediated induction of MnSOD destines cell fate to either G2 arrest or cell death depending on the cellular context. Accordingly, the present study highlights the role of BTG2 as a regulator of ROS level by MnSOD induced via NFκB activation. BTG2-mediated activation of NFκB via PI3K-Akt pathway was independent of p53 status in normal and cancer cells (Figures 4 and 5, and Additional file 2: Figure S2) such as HeLa (p53 low expresser due to HPV E6 & E7 gene expression), A549, wt-MEF and MCF7 cells (wt-p53), therefore, BTG2-regulated crosstalk between PI3K-Akt1 and NFκB pathways was active in a p53-independent manner. Recently, there are several published reports on the crosstalk; in the growth and survival of primary effusion lymphoma cells [40], malignant invasion and metastasis of NSCLC cells [41], and antiproliferative and proapoptotic effects of metformin in various tumor cells [42]. In addition, DHEA-induced proliferation of ventral prostate epithelial cells and T-cell activation are also finely tuned by NFκB activation via PI3K-Akt [43,44]. However, the above reports did not identify any specific transducer between PI3K-Akt1 and NFκB pathways, whereas we presented here BTG2 as a signal mediator between the two pathways via accelerating IκBα degradation. Nevertheless, how BTG2 induces Akt1 activity still needs to be characterized.
The endogenous expression and activity of MnSOD are significantly downregulated in various cancer cells and tumor tissues as compared with counterpart normal cells [45]. Many studies showed that deficiency of the enzyme activity might be due to the reduction of its transcription. In the present study, we presented for the first time precise signal pathways of MnSOD upregulation by BTG2, in addition to transcriptional regulation of MnSOD expression by enhancer activation of the gene in a BTG2-concentration dependant manner (Figure 1), clearly confirming the involvement of BTG2 in the regulation of MnSOD activity in vivo. In the synchronized HeLa cells, BTG2 downregulated cyclin B1 biosynthesis along with G2/M arrest ( Figure 6A-6C). The phenomenon is well accordant with our previous report on the disruption of cyclin B1-FoxM1 regulation loop by BTG2 [12]. In addition, sequential inductions of MnSOD and p21 WAF1 expressions ( Figure 6D and 6E) can also be supported by the notion that NFκB enhances G2/M arrest via induction of p21 WAF1 [38]. Even though p21 WAF1 is a p53 downstream gene, it is also regulated in the p53-independent manner by NFκB activation when the cells are treated with doxorubicin [46], moreover, upstream promoter of p21 WAF1 contains NFκB response elements [47], supporting that NFκB has been considered as a transcription factor for p21 WAF1 independent of p53. Fortuitously, all the available data indicate the BTG2-mediated NFκB activation via its interaction with κB-RE and subsequent upregulation of p21 WAF1 in the p53 nonfunctioning cancer cells.
Depending on the level of ROS, cellular responses are quite variable, ranging from transient to permanent growth arrest, or the cell cycle progression. The series of changes observed in the synchronized HeLa cells (Figure 6), e.g. delayed biosynthesis of cyclin B1 along with delayed entry into G2 phase at 4 h, increase of MnSOD expression at 6 h, significant reduction of H 2 O 2 level at 7 h, and then p21 WAF1 induction and G2/M phase arrest at 8 h after the release from thymidine double blocks reflect a mechanism of APRO activity exhibited by BTG2. Nonetheless, cellular response to BTG2 expression would be diverse, depending on the cellular context and its ROS level.

Conclusions
It is well documented that cancer cells contain defective mitochondria and often reprogram their metabolic pathways to meet with their energy requirements during the process of tumor progression [48]. The major metabolic phenotype described in Warburg effect is the shift of ATP generation from oxidative phosphorylation to glycolysis under physiologic oxygen concentration [49].
Here, MnSOD plays a critical role in the maintenance of mitochondrial integrity, because cells lacking MnSOD are defective in ATP generation by impairing oxidative phosphorylation. Therefore, MnSOD has been regarded as a guardian of powerhouse in tumorigenesis models [50]. Low level of MnSOD may create threat to mitochondrial function at the early stages of carcinogenesis [51], therefore, cancer cells contain defective mitochondria due to lower expression of MnSOD. In that sense, it is worth to note that BTG2-induced MnSOD expression might be one of the early events of tumor suppression by maintaining the integrity of mitochondria. We have recently observed that exogenously expressed BTG2 was rapidly translocated to mitochondria in response to H 2 O 2 in H9c2 cardiomyoblasts [52]. Therefore, we speculate that BTG2 might play a significant role in the modulation of mitochondrial defect in cancer cells. In vivo studies using BTG2 knockout mice might be helpful in finding the significance of BTG2 in carcinogenesis.

Materials and methods
Cell treatment reagents and plasmids

Adenoviral transduction of HeLa cells with BTG2 gene
Ad-BTG2 virus was prepared in our laboratory according to the method described previously [3,12,32] and infected to HeLa cells for 5 h, and then incubated in the complete media for 48 h until subjected to various analyses. Infection of Ad-LacZ was employed as the control of adenoviral transduction.
Cloning of κB-response element (κB-RE) into pGL3 basic vector Cloning of enhancer element of MnSOD gene was performed in our laboratory by PCR amplification using human genomic DNAs isolated from HeLa cells as the template, and the upstream and downstream primers were obtained from the 2 nd intron of MnSOD gene, 5′ ACCTCGAGTGATTGTGTTTGAAGTAAATG-3′ and 5′ AAAAAGCTTTGATTCCACAAGTAAAGG-3′, respectively. PCR amplification was performed using pfu polymerase (Takara Inc., Japan) according to the protocol; Denaturation at 95°C for 30s, annealing at 55°C for 30s, and elongation at 72°C for 60s. The amplified PCR products were digested with XhoI and HindIII, and then subcloned into pGL3 basic luciferase reporter vector purchased from Promega (Madison, WI) using the same sites. The recombinant DNA sequences, κB-RE, were confirmed by nucleotide sequencing (Genotech Corp., Daejeon, Korea).

Transfection analyses
Increasing amounts of BTG2 /TIS21 cDNAs were cotransfected with either promoter construct or κB-RE of MnSOD gene using Metafectane reagent (BionTex, Munich, Germany) and thymidine kinase promoterdriven Renilla luciferase plasmid as a control DNA. Reversal of cross-linking was performed by adding 20 μl of 5 M NaCl to the elution mixture and heating at 65°C overnight. The elutes were mixed with 0.5 M EDTA, 1.0 M Tris (pH 6.5) and 2 μl of 10 mg Proteinase K and shaken well. DNAs recovered by phenol:chloroform extraction and ethanol precipitation were resuspended in nuclease-free water for PCR amplification using the primers described in the Additional file 5.

Measurement of the level of reactive oxygen species
The intracellular H 2 O 2 concentration was measured by FACS analysis using 20 μM 2′,7′-dichlorodihydrofluorescein diacetate (H 2 -DCFDA, Molecular Probes). Cells infected with Ad-BTG2 or Ad-LacZ were pretreated with H 2 -DCFDA for 10 min and then the fluorescence of dichlorodihydrofluorescein derived from oxidation of H 2 -DCFDA was measured.

Small interfering RNAs (siRNAs) and preparation of lentivirus with IκBα mutant
The five sets of siBTG2 sequences and one set of siAtk1 used in the experiments are described in the Additional file 6. The mixture containing siRNAs was prepared with a concentration of 4 μM each and used for transfection of normal and cancer cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Lentivirus with IκBα-mutant was prepared in 293 T cells by cotransfection of IκBα-mutant (Ser32, Ser36 to Ala32, Ala36) and pCMV-VSV-G helper constructs using Lipofectamine 2000. Viral supernatants were harvested in 48 h and filtered through a 0.22 μm filter (Millipore). HeLa cells (2 × 10 5 /60 mm-diameter) were transduced with 250 μl of the supernatant. Culture medium was changed in 12 h and maintained for 2 days until analysis.

RT-PCR
Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume. The gene of interest was amplified by ExTaq polymerase in PCR kits using primer sequences described in the Additional file 5.

Immunoblot analyses
Cells were sonicated with RIPA buffer [50 mM Tris/ HCl, pH 7.5, 150 mM NaCl, 1.0% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 50 mM sodium fluoride, 1.0 mM sodium vanadate, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 μg/ml leupeptin], and 40 μg of cell lysates were resolved on 8 to 12% SDS-PAGE in 25 mM Tris-glycine buffer. The gel-resolved proteins were then transferred to nitrocellulose membrane. The membranes were blocked with 5% nonfat skim milk in PBS containing 0.05% Tween 20 (PBST) for 1 h and then incubated with respective antibodies overnight at 4°C. Nitrocellulose membranes were washed three times with PBST and then incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h. ECL (Amersham Biosciences, UK) kit was employed to visualize protein expression levels. Protein bands were quantified by relative optical densities using Image J software.

Cell synchronization and FACS analysis
HeLa cells were synchronized with 1.0 mM thymidine treatment for 20 h, released for 9 h and then treated again with thymidine for 13 h more before release from the treatment. In the meantime, cells were infected with