romo1 is a negative-feedback regulator of myc · post-translational level through regulation of its...

Author Correction

Romo1 is a negative-feedback regulator of MycSeung Baek Lee, Jung Jin Kim, Jin Sil Chung, Myeong-Sok Lee, Kee-Ho Lee, Byung Soo Kim and Young Do Yoo

Journal of Cell Science 124, 2512 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.095042

There was an error published in J. Cell Sci. 124, 1911-1924.

William P. Tansey should not have been included as an author on this paper. The author list should read: Seung Baek Lee, Jung Jin Kim,Jin Sil Chung, Myeong-Sok Lee, Kee-Ho Lee, Byung Soo Kim and Young Do Yoo.

The authors apologise for this mistake.

Research Article 1911

IntroductionMyc protein levels are increased in response to mitogenic stimulito stimulate G1–S phase progression of the cells, and the expressionlevel of Myc is tightly controlled through transcriptional,translational and post-translational mechanisms (Arnold and Sears,2008). Although Myc transcription is induced during G0–G1transition, fine modulation of the Myc protein level occurs at thepost-translational level through regulation of its stability (Bhatia etal., 1993). The main mechanism for Myc degradation involvesubiquitin-mediated proteolysis (Gross-Mesilaty et al., 1998). Mycis polyubiquitylated by E3 ubiquitin ligases, including the F-boxproteins Fbw7 and Skp2, and a series of sequential phosphorylationevents is required for Fbw7-mediated proteasomal degradation.Phosphorylation sites of Myc include Thr58 and Ser62.Phosphorylation at Ser62 occurs via the Ras–Raf–MEK–ERKpathway (Alvarez et al., 1991; Seth et al., 1991). Ras activationalso inhibits the phosphatidylinositol-3-OH-kinase (PI3K)/Aktpathway to inhibit GSK-3, resulting in stabilization of the Mycprotein (Cross et al., 1995; Sears et al., 2000). During the laterstages of G1, Ras activity decreases, and GSK-3 is reactivated tophosphorylate Myc at Thr58 (Henriksson et al., 1993; Saksela etal., 1992). Myc phosphorylated at Thr58 is ubiquitylated by theSCFFbw7 ubiquitin machinery for degradation by the 26Sproteasome (Welcker et al., 2004; Yada et al., 2004).

Another important mechanism for ubiquitin-mediatedproteasomal degradation of Myc is the Skp2 pathway (Kim et al.,2003; von der Lehr et al., 2003). Skp2 is also reported toubiquitylate Cdk inhibitors and tumor suppressor proteins such asp27Kip1 (Carrano et al., 1999), p57Kip2 (Kamura et al., 2003), p130(Tedesco et al., 2002) and Tob1 (Hiramatsu et al., 2006). Skp2 isoverexpressed in cancer (Bashir and Pagano, 2003). Theexpressions of Skp2 and Myc are induced by mitogenic stimulation;

however, Skp2 expression continues into S phase (Lisztwan et al.,1998). Skp2 interacts with two domains of Myc (residues 129–147: the N-terminal Myc box II domain and residues 379–418: theC-terminal bHLHZip domain) at the G1 to S phase transition toinduce Myc degradation and turnover (Kim et al., 2003; von derLehr et al., 2003). Skp2-mediated ubiquitylation does not correlatewith Myc phosphorylation because Myc mutated at Thr58 is wellubiquitylated (Kim et al., 2003).

Romo1 (reactive oxygen species modulator 1) was first identifiedin 2006, and forced expression of Romo1 increases the level ofcellular ROS that originate from mitochondria (Chung et al., 2006).Romo1 is localized to the mitochondria and releases mitochondrialROS through complex III of the mitochondrial electron transportchain (Chung et al., 2008). Although the ROS produced by cytosolicenzymes such as NADPH oxidase have a role in cell proliferation,mitochondrial ROS are not known to be involved in cellproliferation. Recently, we reported that ROS originating from theendogenous Romo1 protein are necessary for both normal andcancer cell proliferation (Chung et al., 2009; Na et al., 2008).Suppression of Romo1 expression inhibits cell growth throughinhibition of ERK activation and p27Kip1 expression, demonstratingthat ROS derived from Romo1 are required for cell proliferation.Romo1 expression is enhanced in senescent cells and in mostcancer cells (Chung et al., 2006; Chung et al., 2008). Romo1 isalso upregulated by serum deprivation and contributes to the serum-deprivation-mediated increase in ROS (Lee et al., 2010).Furthermore, a recent paper demonstrated that Romo1 modulatesROS production in the mitochondria (Kim et al., 2010). Romo1recruits the anti-apoptosis regulator Bcl-XL to decrease themitochondrial membrane potential in response to tumor necrosisfactor- (TNF-), resulting in ROS production. Although manystudies have been conducted on Romo1, its physiological function

SummaryDegradation of Myc protein is mediated by E3 ubiquitin ligases, including SCFFbw7 and SCFSkp2, but much remains unknown aboutthe mechanism of S-phase kinase-associated protein (Skp2)-mediated Myc degradation. In the present study, we show that upregulatedMyc protein, which triggers the G1–S phase progression in response to growth-stimulatory signals, induces reactive oxygen speciesmodulator 1 (Romo1) expression. Romo1 subsequently triggers Skp2-mediated ubiquitylation and degradation of Myc by a mechanismnot previously reported in normal lung fibroblasts. We also show that reactive oxygen species (ROS) derived from steady-state Romo1expression are necessary for cell cycle entry of quiescent cells. From this study, we suggest that the generation of ROS mediated bypre-existing Romo1 protein is required for Myc induction. Meanwhile, Romo1 expression induced by Myc during G1 phase stimulatesSkp2-mediated Myc degradation in a negative-feedback mechanism.

Key words: Myc, Romo1, ROS, Skp2

Accepted 18 January 2011Journal of Cell Science 124, 1911-1924 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.079996

Romo1 is a negative-feedback regulator of MycSeung Baek Lee1, Jung Jin Kim1, Jin Sil Chung1, Myeong-Sok Lee2, Kee-Ho Lee3, Byung Soo Kim4, William P. Tansey5 and Young Do Yoo1,*1Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705,Republic of Korea2Department of Biological Sciences, Sookmyung Women’s University, Seoul 140-742, Republic of Korea3Department of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea4Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705, Republic of Korea5Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA*Author for correspondence ([email protected])

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is not well elucidated. In the present study, we investigated the roleof Myc-induced Romo1 in Myc turnover after serum stimulation.

ResultsMyc expression induced after serum stimulation increasesRomo1 expressionMyc has been reported to stimulate ROS generation, which in turninduces DNA damage (Vafa et al., 2002). Romo1 has been alsoreported to recruit Bcl-XL to reduce the mitochondrial membranepotential in response to TNF-, resulting in mitochondrial ROSgeneration (Chung et al., 2006; Kim et al., 2010). Therefore, weinvestigated the correlation between Myc and Romo1 after serumstimulation. The expressions of Myc, Romo1 and p27Kip1

(CDKN1B) were examined after addition of serum to cultures ofnormal human lung fibroblasts (IMR-90 and WI-38 cells) andhuman embryo kidney cells (HEK293). Low basal levels of Mycand Romo1 and high levels of p27Kip1 were detected in unstimulatedcells by western blot analysis (Fig. 1A). When the cells werestimulated with serum, Myc expression was induced at 1 hour andits level peaked at 3–6 hours after serum treatment. Interestingly,Romo1 expression was enhanced after Myc induction, peaking at9–24 hours (Fig. 1A). By contrast, p27Kip1 was downregulated at6 hours after serum stimulation.

To observe whether Myc induced Romo1 expression, HEK293and HeLa cells were transfected with Myc, and Romo1 expressionwas observed by western blot analysis. As shown in Fig. 1B, Myc

1912 Journal of Cell Science 124 (11)

Fig. 1. Myc-induced Romo1 expressionafter serum stimulation. (A)Myc, Romo1and p27Kip1 expression levels after serumstimulation were examined by western blotanalysis in IMR-90, WI-38 and HEK293cells. The cells were serum-starved for 48hours and then treated with 30% serum. -actin was used as a loading control. (B)Mycinduces Romo1 expression. HEK293 andHeLa cells were transfected with Myc orvector alone and western blot analysis ofRomo1 was performed at the indicatedtimes. (C,D)After the cells were transfectedwith MYC siRNA and were serum-starvedfor 48 hours, serum was added to the cellsand western blot analyses of Myc (C) andRomo1 (D) were performed. (E)ROMO1mRNA induction after serum stimulation.After HEK293 cells were serum-starved,semi-quantitative RT-PCR (upper panel) andreal time-PCR (lower panel) analyses wereperformed. Results represent the means (± s.e.m.) of three independent experimentsperformed in triplicate. The relativeinduction of ROMO1 mRNA wasnormalized to ACTB (-actin) or GAPDH.

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increased the Romo1 protein level, demonstrating that Romo1 isdownstream of Myc. Next, we examined whether serum-stimulatedMyc expression also increased Romo1 expression. To observewhether knockdown of Myc blocked serum-induced Romo1expression, WI-38 cells and IMR-90 cells were transfected withMYC siRNA. MYC siRNA transfection efficiently inhibited Mycinduction by serum stimulation (Fig. 1C), and Myc knockdownblocked the serum-induced Romo1 expression (Fig. 1D).Furthermore, semi-quantitative RT-PCR and real-time PCR analysesdemonstrated that serum stimulation upregulated ROMO1expression transcriptionally (Fig. 1E). These results demonstratethat serum stimulation enhances Myc-mediated Romo1 expression.

Romo1 regulates serum-induced ROS generation and Mycprotein levelTo investigate whether knockdown of Romo1 suppressed serum-induced ROS generation, IMR-90 cells were transfected withROMO1 siRNA and ROS levels were measured by staining thecells with MitoSOX, a probe for superoxide in the mitochondria.First, Romo1 knockdown in cells transfected with ROMO1 siRNAwas examined by western blot analysis (supplementary materialFig. S1A). To exclude off-target effects of the ROMO1 siRNAconstruct, a rescue experiment was performed. The N-terminaldeletion mutant of Romo1 (Romo1-N), which does not includethe ROMO1 siRNA-1 sequence, is known to induce mitochondrialROS generation (Kim et al., 2010). ROMO1 siRNA-1 transfectionefficiently caused Romo1 knockdown and decreased ROS levelsin various cell lines (Hwang et al., 2007; Na et al., 2008). However,Romo1-N was resistant to ROMO1 siRNA-1 (supplementarymaterial Fig. S1B). In the present study, we showed that both wild-type Romo1 and Romo1-N decreased Myc expression, butRomo1-C did not (Fig. 4D). Therefore, we examined whetherRomo1-N can downregulate Myc expression in cells transfectedwith ROMO1 siRNA-1. As shown in supplementary material Fig.S1C, FLAG–Romo1 (wt) decreased Myc expression and ROMO1siRNA-1 transfection blocked Romo1-induced Mycdownregulation. However, ROMO1 siRNA-1 transfection failed tosuppress Romo1-N-induced Myc downregulation. This resultshowed the specificity of the ROMO1 siRNA construct. Next, weexamined whether Romo1 knockdown suppressed serum-inducedROS production. As shown in Fig. 2A, high ROS levels wereobserved in serum-deprived cells and Romo1 knockdown inhibitedserum deprivation-induced ROS production. The ROS increaseswere suppressed by serum addition. These results are consistentwith a previous report (Lee et al., 2010). In Fig. 1A, we showedthat serum stimulation increased Romo1 expression. This Romo1induction was also observed by fluorescence microscopy (Fig.2A). The serum-stimulated ROS increases were completely blockedby Romo1 knockdown (Fig. 2A). The decreases in Romo1expression and ROS formation in primary human fibroblasts IMR-90 (Fig. 2B,C) or WI-38 (supplementary material Fig. S2A) werequantified using MetaMorph software. In Fig. 1D, we showed thatknockdown of Myc blocked the serum-induced Romo1 expression.Therefore, we also measured the ROS formation after serumstimulation in cells transfected with MYC siRNA. As shown insupplementary material Fig. S2B, the serum-stimulated ROSincreases were also blocked by Myc knockdown. Next, weexamined Myc expression in IMR-90, WI-38 and HEK293 cellstransfected with ROMO1 siRNA. Interestingly, Romo1 knockdownblocked the elimination of Myc after 6 hours of serum stimulation(Fig. 2D,E). Instead, Myc expression was gradually increased until

24 hours. From this result, we suggest that the Romo1 expressioninduced by Myc during G1 phase is necessary for elimination ofMyc and we assume that increased Romo1 expression might beinvolved in Myc degradation in a negative-feedback mechanism.

Although Myc expression was increased in cells transfectedwith ROMO1 siRNA after 6 hours of serum stimulation comparedwith control cells, its expression was very low at early times aftermitogenic stimulation (0–3 hours, Fig. 2D). Recently, we reportedthat ROS derived from Romo1 expression also regulate cellproliferation through activation of ERK in various normal andcancer cell lines (Chung et al., 2009; Na et al., 2008). Therefore,we examined whether ROS derived from Romo1 expression wererequired for induction of Myc in early G1 phase. As shown in Fig.3A, Myc induction for 2 hours after serum stimulation wassuppressed by Romo1 knockdown. Treatment with antioxidantsalso inhibited Myc induction. However, hydrogen peroxide (H2O2)treatment of cells transfected with ROMO1 siRNA recovered Mycexpression (Fig. 3B). Myc expression was also examined usingvarious kinase inhibitors. MEK1/2-specific inhibitors, PD98059and U0126, blocked Myc induction and ERK activation (Fig. 3B).These results demonstrate that ROS derived from Romo1 inresponse to serum stimulation are necessary for Myc induction andcell cycle progression.

To further investigate the correlation between Romo1 expressionand cell cycle transition triggered by serum stimulation, flowcytometric analysis was carried out in IMR-90 cells transfectedwith ROMO1 siRNA. In this experiment, Romo1 knockdowndelayed cell cycle progression into S phase (Fig. 3C). This findingwas also confirmed in WI-38 cells (supplementary material Fig.S2C). These results indicate that Romo1 expression has animportant role in cell cycle entry triggered by mitogenic stimulationvia ERK activation and Myc induction. We also suggest that thebasal level of ROS derived from the steady-state level of Romo1is required for ERK activation and Myc stabilization. By contrast,enhanced ROS levels generated from Romo1 expression, which isinduced by Myc, trigger the elimination of Myc.

Romo1 expression induces Myc degradationAs shown in Fig. 2D, knockdown of Romo1 blocked theelimination of Myc. Therefore, we investigated whether increasedRomo1 expression caused Myc downregulation. Romo1 wastransfected into HeLa cells, and Myc expression was measured bywestern blot and immunofluorescence analysis. Romo1overexpression triggered downregulation of Myc (Fig. 4A andsupplementary material Fig. S3A). To confirm this finding, cellswere co-transfected with Myc and Romo1, and Myc expressionwas again measured by western blot analysis. As shown in Fig. 4B,Romo1 also induced the downregulation of Myc that was expressedexogenously. Expression of Romo1 also decreased the expressionof Myc in Huh-7, HeLa, A549 and H1299 cells (supplementarymaterial Fig. S3B).

Next, we analyzed which domain of Myc was responsible for itsexpression. Romo1 and Myc deletion constructs (Herbst et al., 2004;Tworkowski et al., 2002) were co-transfected into the cells, and Mycexpression was measured by western blot analysis. We found thatRomo1 promoted the downregulation of wild-type (WT) Myc, a Aconstruct including Myc box (Mb) I, a B construct, a E constructand a G construct. However, Romo1 failed to downregulate a Cconstruct including the Mb II domain, a D construct including thePEST domain, and a F construct (Fig. 4C). This result demonstratesthat the Mb I domain, which is needed for Fbw7-mediated

1913Myc regulation by Romo1

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proteasomal degradation of Myc, is not required for Myc degradationand that another mechanism exists for ubiquitin-mediatedproteasomal degradation of Myc. Recently, we reported that the C-terminal region of Romo1 is important for TNF--induced ROSproduction (Kim et al., 2010). To examine the effects of Romo1deletion constructs on Myc expression, two deletion constructs ofRomo1, designated FLAG–Romo1-C (deletion of the C-terminal

48–79 residues) and FLAG–Romo1-N (deletion of the N-terminal1–16 residues), were transfected into HeLa cells and Myc expressionwas assessed. As shown in Fig. 4D, both wild-type Romo1 andRomo1-N decreased Myc expression, but Romo1-C did not.From this result, we suggest that ROS derived from the C-terminaldomain of Romo1 induced Myc degradation through the Mb IIdomain, Mb III domain and a F construct (316–378 residues).


Fig. 2. Blockage of serum-induced ROS production andMyc elimination byknockdown of Romo1.(A)Serum-induced ROSproduction is blocked byROMO1 siRNA transfection.After transfection with ROMO1siRNA, IMR-90 cells wereserum-starved for 48 hours andthen treated with serum. Thecells were stained withMitoSOX for 30 minutes andthen observed by fluorescencemicroscopy. (B)Forquantification purposes, theimages were overlaid, andRomo1 expression (green) wasanalyzed with MetaMorphsoftware. Results represent themeans (± s.e.m.) of threeindependent experimentsperformed in triplicate. *P<0.05versus control siRNA; #P<0.05and ##P<0.01 versus controlsiRNA at 0 hours by two-wayANOVA. (C)For quantificationpurposes, the images wereoverlaid by a computer, andMitoSOX fluorescence (red)was analyzed with MetaMorphsoftware. Results represent themeans (± s.e.m.) of threeindependent experimentsperformed in triplicate.*P<0.05; **P<0.01 versuscontrol siRNA; #P<0.05 versuscontrol siRNA at 1 hour by two-way ANOVA. (D)Aftertransfection with ROMO1siRNA, IMR-90, WI-38 andHEK293 cells were serum-starved for 48 hours and thentreated with serum. Mycexpression was measured bywestern blot analysis at theindicated times. (E)Theintensity of Myc expression inIMR-90 cells was quantified byscanning densitometry. Resultsrepresent the means (± s.e.m.)of three independentexperiments performed intriplicate. **P<0.01;***P<0.001 versus controlsiRNA by one-way ANOVA.

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Romo1 is localized in the mitochondria and inducesmitochondrial ROS production through complex III of themitochondrial electron transport chain (Chung et al., 2008). Todetermine whether mitochondrial ROS production through complexIII is required for downregulation of Myc, HeLa cells were culturedin the presence of an antioxidant (trolox), mitochondrial respiratorychain complex III inhibitors (myxothiazol and stigmatellin),complex I inhibitor (rotenone), complex II inhibitor (malonate) orcomplex IV inhibitor (sodium azide), and Myc downregulationwas assessed by western blot analysis (Fig. 4E). Romo1-triggereddownregulation of Myc was blocked by myxothiazol andstigmatellin and by trolox. By contrast, the other inhibitors failedto inhibit Romo1-mediated Myc downregulation. Next, we treatedthe cells with increasing amounts of H2O2, and Myc downregulationwas analyzed by western blot analysis. As shown in Fig. 4F,treatment with a low concentration of H2O2 increased the amountof Myc protein. By contrast, treatment with higher concentrationsof H2O2 decreased the amount of Myc protein. These findingsindicate that Romo1-derived ROS have an important role in Mycregulation.

Romo1 induces Myc degradation through cytoplasmictranslocation of Skp2Myc degradation is regulated by Fbw7 and Skp2, and Mycdegradation by Fbw7 is dependent on the phosphorylation of Thr58and Ser62 in the MB1 domain (Welcker et al., 2004; Yada et al.,2004). To ascertain whether Myc degradation controlled by Romo1

is related to the phosphorylation of Thr58 and Ser62, wild-type(WT) Myc or Myc mutants (T58A, S62A or T58AS62A) weretransfected into HeLa cells, and Myc expression was examined bywestern blot analysis. As shown in Fig. 5A, Myc expression wasdecreased in cells expressing Romo1, Skp2 or Fbw7. As expected,Fbw7 expression failed to degrade the Myc protein in cellstransfected with Myc mutants (Fig. 5B). This result is consistentwith a previous report (Welcker et al., 2004; Yada et al., 2004).However, Romo1 and Skp2 efficiently degraded the Myc proteinin cells transfected with Myc mutants, demonstrating that Romo1-triggered Myc degradation is not mediated by Fbw7 (Fig. 5B,C).H2O2 treatment also efficiently triggered Myc degradation in cellstransfected with Myc mutants (Fig. 5D). Next, we investigatedwhether Romo1 stimulated Myc degradation through Skp2. SKP2siRNA was transfected into cells to knock down Skp2(supplementary material Fig. S4) and Myc expression wasexamined. Interestingly, Skp2 knockdown suppressed Romo1-induced Myc degradation (Fig. 5E).

Recent reports have shown that the phosphorylation of Skp2 atSer72 by Akt leads to cytoplasmic translocation of Skp2 (Gao etal., 2009; Lin et al., 2009). To investigate whether Romo1expression regulates Skp2 cytoplasmic translocation, we observedHeLa cells by fluorescence microscopy after Romo1 transfection.As shown in Fig. 6A, Romo1 expression induced the cytoplasmictranslocation of Skp2. Cytoplasmic Skp2 levels were quantified byfluorescence microscopy and analysis with MetaMorph software.This finding was also confirmed in HEK293 cells (supplementary


Fig. 3. ROS derived from Romo1 regulateMyc induction through Erk activation forcell cycle entry. (A)After HEK293 cells weretransfected with ROMO1 siRNA and serum-starved for 48 hours, the cells were treated withserum to induce cell cycle entry. Western blotanalysis was performed using antibodiesagainst the indicated proteins. (B)AfterHEK293 cells were treated with MEK1/2-specific inhibitors (25M PD98059 and 1 mMU0126), PI3K-specific inhibitors (20MLY294002 and 1M wortmannin), JNKinhibitor (20M SP600215), p38 kinaseinhibitor (25M SB203580), GSK-3 inhibitor(25M TWS119), antioxidants (1 mM NACand 1M trolox) or H2O2 (10M), westernblot analysis was performed using antibodiesagainst the indicated proteins. (C)After IMR-90 cells were treated with ROMO1 siRNA andserum-starved for 48 hours, the cells weretreated with serum and analyzed by flowcytometry.

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material Fig. S5A). To determine whether H2O2 treatment also ledto the cytoplasmic translocation of Skp2, the cells were treatedwith H2O2. H2O2 treatment promoted the cytoplasmic translocationof Skp2 (Fig. 6B), which was confirmed in cells exogenouslytransfected with Skp2 (Fig. 6C). We also confirmed the localizationof Skp2 by cellular fractionation of HeLa cells. The cells weretreated with H2O2 or transfected with FLAG–Romo1. Both Romo1and H2O2 induced cytoplasmic translocation of Skp2 (Fig. 6D).These results demonstrate that ROS derived from Romo1 promotethe cytoplasmic translocation of Skp2.

Next, we performed an immunofluorescence assay to detect thesubcellular localization of Skp2 in response to serum stimulationin normal human fibroblasts, IMR-90 cells. The cells were serum-starved for 48 hours and then treated with serum for 15 hours. Thecells were then harvested for immunofluorescence analysis. Skp2was observed in the nucleus before serum stimulation. However,Skp2 was translocated into cytoplasm after serum addition for 15

hours (Fig. 6E). A similar finding was also observed in othernormal human fibroblasts, WI-38 cells (supplementary materialFig. S5B).

We showed that downregulation of Myc by Romo1 is requiredfor mitochondrial ROS production through complex III of themitochondrial electron transport chain (Fig. 4E). Therefore, weexplored whether cytoplasmic translocation of Skp2 by Romo1was blocked by inhibitors of complex III of the mitochondrialelectron transport chain. After HeLa cells were transfected withFLAG–Romo1, the cells were incubated with various inhibitors ofthe mitochondrial electron transport chain. Cytoplasmictranslocation of Skp2 was detected in cells transfected with Romo1(Fig. 6F). However, the Romo1-induced Skp2 cytoplasmictranslocation was inhibited by myxothiazol and stigmatellin, butwas not affected by other inhibitors. Trolox was used as a positivecontrol. We also investigated whether Romo1 expression regulatedcytoplasmic translocation of Myc in HeLa cells after Romo1


Fig. 4. ROS derived from Romo1 stimulate downregulation of Myc. (A)Romo1 expression induces Myc downregulation. HeLa cells were transfected withFLAG–Romo1, and Myc expression was examined by western blot analysis. (B)WI-13 VA13 and HEK293 cells were transfected with Myc or FLAG–Romo1, andMyc expression was examined by western blot analysis. (C)Schematic representation of the structural organization of Myc (top). Myc deletion constructs weretransfected into HeLa cells and Myc expression was examined by western blot analysis (bottom). TAD, transactivation domain; Mb, myc box; NLS, nuclearlocalization sequence; BR, basic region; HLH, helix-loop-helix motif; Zip, leucine zipper motif. (D)Schematic representation of the structural organization ofRomo1 (top). Romo1 deletion constructs (C or N) were transfected into HeLa cells and Myc expression was examined by western blot analysis (bottom). TM,transmembrane domain; Wt, Romo1 wild-type; C, Romo1 C-terminus deletion; N, Romo1 N-terminus deletion. (E)After HeLa cells were transfected with Mycor FLAG–Romo1, cells were incubated with trolox (1 or 10M), myxothiazol (1 or 10M), stigmatellin (1 or 10M), rotenone (1 or 10M), malonate (10 or100M) or sodium azide (1 or 10 mM) for 4 hours. Myc expression was examined by western blot analysis. (F)HEK293, WI-38 VA13 and H1299 cells weretreated with increasing amounts of H2O2 (1–1,000M) and Myc expression was examined by western blot analysis.

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transfection. Romo1 expression enhanced the cytoplasmictranslocation of Myc (Fig. 6G). We also confirmed the localizationof Myc by cellular fractionation of HeLa cells transfected withFLAG–Romo1 (Fig. 6H).

To investigate whether Romo1 promotes the cytoplasmictranslocation of Skp2 through the PI3K–Akt pathway, HeLa cellswere transfected with FLAG–Romo1. As shown in Fig. 7A, Romo1expression triggered Akt activation and Myc degradation. The Aktactivation and Myc degradation were inhibited by the PI3K inhibitorLY294002. Immunofluorescence experiments revealed that Romo1expression induced the cytoplasmic translocation of Myc and Skp2,and their translocations were suppressed by LY294002 (Fig. 7B).We further investigated whether the H2O2-induced cytoplasmictranslocation of Skp2 was blocked by LY294002.Immunofluorescence analysis showed that cytoplasmictranslocation of exogenous Skp2 by H2O2 treatment was inhibitedby LY294002 and trolox (Fig. 7C). These results suggest that thecytoplasmic translocation of Skp2 and Myc induced by Romo1 ismediated by Akt.

Romo1 enhances the interaction between Skp2 and Mycand Myc ubiquitylationTo determine whether Romo1 expression enhances the interactionbetween Skp2 and Myc, FLAG–Romo1 was transfected into HeLacells and a co-immunoprecipitation experiment was performed. Asshown in Fig. 8A, Romo1 expression increased Skp2 binding toMyc and trolox treatment inhibited this interaction. To confirm thisinteraction, Skp2 was immunoprecipitated with its antibody andwestern blot analysis was performed with anti-Myc antibody (Fig.

8B). Next, we examined whether Romo1 enhanced Mycubiquitylation. Myc was transfected into HeLa cells, together withFLAG–Romo1, FLAG–Fbw7 or FLAG–Skp2. To identify theextent of Myc ubiquitylation, ubiquitylated-Myc wasimmunoprecipitated with anti-HA antibody and subjected to westernblot analysis using anti-Myc antibody. As shown in Fig. 8C,D,Romo1 significantly increased the amount of Myc-ubiquitinconjugates, and trolox treatment suppressed Myc ubiquitylation.Fbw7 and Skp2 were used as positive controls. Fig. 8E showedthat Romo1-N significantly increased the amount of Myc-ubiquitin conjugates, but Romo1-C had no effect on Mycubiquitylation. This finding was also examined in HEK 293 cells(supplementary material Fig. S6). We also explored whether H2O2

enhances ubiquitylation of Myc (Fig. 8F). These results indicatethat ROS increase modulated by Romo1 expression induces aninteraction between Skp2 and Myc and then enhances Mycubiquitylation. To identify whether Romo1 regulates the stabilityof Myc protein, Romo1 was expressed by transient transfection inHeLa cells and the cells were treated with cycloheximide (CHX).The half-life of Myc was decreased in cells transfected with Romo1(Fig. 8G, upper panel). Romo1 also reduced the stability ofexogenously transfected Myc in the cells (Fig. 8G, lower panel).These results suggest that Romo1 negatively controls Myc stabilityand that it is an important post-translational regulator of Mycexpression.

DiscussionMyc is an unstable protein with a half-life of 20–30 minutes (Hannand Eisenman, 1984) and Myc degradation during G1-S phase


Fig. 5. ROS derived from Romo1trigger Myc degradation throughSkp2. (A)HeLa cells were co-transfected with Myc, FLAG–Skp2,FLAG–Fbw7 or FLAG–Romo1.Myc expression was examined bywestern blot analysis. (B)AfterHeLa cells were co-transfected withFLAG–Myc (Wt, a Thr58 mutant, aSer62 mutant or a Thr58,Ser62mutant of Myc), HA–Fbw7 orFLAG–Skp2, Myc expression wasexamined by western blot analysis.(C)Romo1 induces the Thr58- orSer62-phosphorylation-independentdegradation of Myc. HeLa cellswere co-transfected with FLAG–Romo1 and FLAG–Myc (Wt, T58A,S62A or T58AS62A). Mycexpression was examined by westernblot analysis. Asterisk, non-specificband. (D)ROS trigger Thr58 orSer62 phosphorylation-independentdegradation of Myc. After HeLacells were transfected with FLAG–Myc (Wt, a Thr58 mutant, a Ser62mutant or a Thr58,Ser62 mutant ofMyc), cells were treated with H2O2

(100M) for 2 hours. (E)Aftertransfection of SKP2 siRNA intoHeLa cells for 24 hours, cells weretransfected with HA–Myc andFLAG–Romo1.

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progression has been well identified. Serum stimulation enhancesRas activation in the PI3K–Akt pathway during early G1, resultingin GSK-3 inhibition (Gregory and Hann, 2000; Sears et al., 2000).During late G1 phase, the Ras activity decreases and GSK-3phosphorylates Myc on Thr 58, resulting in its ubiquitylation anddegradation (Sears et al., 2000). Phosphorylation of Myc on Thr58 plays a key role in Myc degradation, and a mutation on Thr 58contributes to tumorigenesis (Bhatia et al., 1993). However, Mycdegradation by GSK-3-mediated Myc phosphorylation on Thr 58is not sufficient for Myc degradation during late G1 phase. Indeed,Myc mutated at Thr 58 was reported to have a half-life of 30–40minutes, compared to the 20–30 minutes half-life of wild-typeMyc (Bader et al., 1986; Sears et al., 1999; Salghetti et al., 1999).Moreover, Myc mutated at Thr 58 is still targeted for ubiquitylationand degradation (Hann, 2006). Therefore, an additional pathway

should exist for Myc degradation. In the present study, we showedthat Myc expression reached a peak at 3–6 hours and declined at9 hours. However, Myc expression levels continuously increaseduntil 24 hours when Romo1 expression was suppressed (Fig. 2D).We also showed that Romo1 expression promoted the ubiquitylationand degradation of Myc through cytoplasmic translocation of Skp2and Myc (Figs 6 and 8). Therefore, we suggest that theRomo1/ROS/Skp2 pathway is another pathway for Myc turnover.The Romo1-mediated pathway appears to be one of the mainpathways for Myc degradation, because the Myc level wassignificantly decreased when the cells were transfected with Romo1to enhance Romo1 expression (Fig. 4).

Two main pathways of ubiquitin-mediated degradation of Mycexist for Myc turnover. One is mediated by Fbw7. The otherpathway is mediated by Skp2. Myc ubiquitylation through Fbw7


Fig. 6. See next page for legend.

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has been well elucidated. A series of sequential phosphorylationevents occur after mitogenic stimulation and are followed by Fbw7-mediated degradation of Myc (Sears et al., 1999; Yeh et al., 2004).

However, Skp2-mediated Myc degradation is not well understood.In the present study, we demonstrate that Romo1 induces Mycdegradation through a novel mechanism not previously reported.


Fig. 6. Romo1 regulates cytoplasmictranslocation of Skp2.(A)Immunofluorescence staining of HeLacells transfected with FLAG–Romo1. Thedata represent the average of threeexperiments and 150 cells were monitoredin each experiment. Scale bar: 20m.(B)After HeLa cells were incubated in thepresence of H2O2 (200M) for 2 hours,cells were stained as indicated. Cells (150–200) were scored and a representativeresult from three independent experimentsis shown. Scale bar: 20m.(C)Immunofluorescence staining of HeLacells transfected with FLAG–Skp2. Thecells were treated with H2O2 (200M) for2 hours, then fixed and stained asindicated. The data represent the average ofthree experiments and 150 cells weremonitored in each experiment. Scale bar:20m. (D)Western blot analysis of nuclear(N) and cytoplasmic (C) fractions of HeLacells treated with H2O2 (200M) for 2hours or transfected with FLAG–taggedRomo1 for 48 hours. Cell lysates weresubjected to western blot analysis withantibodies against Skp2, FLAG (Romo1),-actin (cytosolic marker) or lamin B1(nuclear marker). (E)IMR-90 cells wereserum-starved for 48 hours and then treatedwith serum for 15 hours. Cells wereharvested for immunofluorescenceanalysis. Cells (150–200) were scored anda representative result from threeindependent experiments is shown. Scalebar: 20m. (F)After HeLa cells weretransfected with FLAG–Romo1, the cellswere cultured in the presence ofmyxothiazol (10M), stigmatellin(10M), rotenone (10M), malonate(100M), sodium azide (10 mM) or trolox(10M) for 4 hours, and harvested forimmunofluorescence analysis. The datarepresent the average of three experiments;100 cells were monitored in eachexperiment. Scale bar: 20m. (G)HeLacells were transfected with the indicatedplasmids, treated with MG132 for 6 hours.Scale bar: 20m. Arrow indicates cellsexpressing FLAG–Romo1; asterisksindicate cytoplasmic Myc protein.(H)Western blot analysis of nuclear (N)and cytoplasmic (C) fractions of HeLacells transfected with FLAG–Romo1 for48 hours. Cell lysates were subjected towestern blot analysis with antibodiesagainst Myc, Flag (Romo1), -actin(cytosolic marker) or lamin B1 (nuclearmarker).

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Romo1 induced by the enhanced Myc level increased the cellularROS level to trigger the cytoplasmic translocation of Skp2 (Fig.6). Skp2 was induced by mitogenic stimulation and reached a peakin S phase. Skp2 binds to two domains of Myc (Kim et al., 2003;von der Lehr et al., 2003). It is unlikely that Romo1 induced Skp2expression because increased Romo1 expression did not increasethe Skp2 level (Fig. 8A). Instead, Romo1 contributed to thecytoplasmic translocation of Skp2. Skp2 is reportedly located inthe nucleus (Miura et al., 1999). However, a recent report showedthat Skp2 translocates into the cytoplasm after Akt-mediatedphosphorylation of Ser 72 (Gao et al., 2009; Lin et al., 2009).Romo1 also regulated the cytoplasmic translocation of Myc in thepresence of the proteosomal inhibitor, MG-132 (Fig. 6G,H). Inaddition to enhancing the cytoplasmic translocation of Skp2 andMyc, Romo1 promoted the interaction between Skp2 and Myc,resulting in Myc ubiquitylation (Fig. 8). It seems that Romo1 doesnot directly interact with Skp2 or Myc in Myc degradation becauseH2O2 treatment increased the cytoplasmic translocation of Skp2and Myc (Fig. 6B). Antioxidant treatment also suppressed thecytoplasmic translocation of Skp2 (Fig. 6F). Previously, we reportedthat ROS originate from complex III of the mitochondrialrespiratory chain (Chung et al., 2008). Therefore, we examinedwhether cytoplasmic translocation of Skp2 was blocked bymitochondrial complex III inhibitors, and we showed that treatmentof complex III inhibitors efficiently suppressed the cytoplasmictranslocation of Skp2 (Fig. 6F). From these results, we suggest that

ROS derived from Romo1 play an important role in Myc turnover.Although we showed that Myc degradation occurs via Romo1-mediated cytoplasmic translocation of Skp2, the exact mechanismby which Romo1 regulates the cytoplasmic translocation of Skp2remains to be studied in the future.

Appropriate ROS levels maintained inside the cells play animportant role in cell growth and survival, and the physiologicalrange of H2O2 concentrations is 0.001 to 0.7 M (Burdon andRice-Evans, 1989). Although excessive ROS production reportedlycontributes to many pathological disorders, including cancer, aging,and neurological diseases, ROS are required for redox signalingand their main source is NADPH oxidase (Finkel, 2003; Turrens,2003). This enzyme responds to growth or cell survival signals toinduce ROS production and is subsequently eliminated byantioxidant enzymes. Although the increase in ROS triggered byRomo1, which is induced by Myc, contributes to Myc degradation,appropriate levels of ROS are critical for Myc stabilization. Theresults presented in this study are consistent with a previous reportimplicating ROS produced by hematopoietic cytokines in G1 to Sprogression in Myc stabilization (Iiyama et al., 2006). This previousstudy also showed that NAC treatment reduced the stability ofMyc protein, while H2O2 treatment of the cells enhanced itsstability. H2O2 treatment induced ERK-dependent Mycphosphorylation on Ser 62 (Benassi et al., 2006). Recently, wereported that Romo1 is necessary for cell growth and that Romo1knockdown induces cell cycle arrest at G1 through inhibition of


Fig. 7. Romo1-induced cytoplasmic translocation of Skp2 and Myc through the PI3K–Akt pathway.(A)HeLa cells were transfected with FLAG–Romo1 and treated with LY294002 (20M) or trolox(1M) for 6 hours before western blot analysis. (B)After HeLa cells were transfected with FLAG–Romo1, the cells were treated with MG132 (10M), LY294002 (20M), then immunofluorescenceanalysis was performed. Scale bar: 20m. (C)Immunofluorescence staining of HeLa cells transfectedwith FLAG–Skp2. The cells were treated with H2O2 (200M) for 2 hours in the presence of LY294002or trolox.

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Fig. 8. Romo1-inducedinteraction of Myc with Skp2and Myc ubiquitylation.(A)After HeLa cells weretransfected with FLAG–Romo1and treated with MG132 (10M)or trolox (1M) for 6 hours,Myc was immunoprecipitatedwith anti-Myc antibody forwestern blot analysis. WB,western blot analysis; WCL,whole cell lysates. (B)AfterHeLa cells were transfected withFLAG–Romo1, Skp2 wasimmunoprecipitated with anti-Skp2 antibody for western blotanalysis. IgG HC,immunoglobulin heavy chain.(C)After HeLa cells weretransfected with Myc, Ubiquitin-HA, FLAG–Romo1, FLAG–Fbw7 or FLAG–Skp2, the cellswere treated with MG132.Ubiquitylated proteins wereimmunoprecipitated with anti-HA antibody for western blotanalysis with anti-Myc antibody.(D)After HeLa cells weretransfected with Myc, Ubiquitin–HA, or FLAG–Romo1, the cellswere treated with MG132 ortrolox. Ubiquitylated proteinswere immunoprecipitated withanti-HA antibody for westernblot analysis with anti-Mycantibody. (E)After HeLa cellswere transfected with Myc,Ubiquitin-HA, FLAG–Romo1(Wt), FLAG–Romo1 (C) orFLAG–Romo1 (N), the cellswere treated with MG132.Ubiquitylated proteins wereimmunoprecipitated with anti-HA antibody for western blotanalysis with anti-Myc antibody.(F)After HeLa cells weretransfected with Myc, Ubiquitin–HA, FLAG–Romo1 and FLAG–Skp2, the cells were treated withMG132 and H2O2 (200M, 2hours). Ubiquitylated proteinswere immunoprecipitated withanti-HA antibody for westernblot analysis with anti-Mycantibody. (G)After HeLa cellswere transfected with FLAG–Romo1 (upper panel) or co-transfected with Myc andFLAG–Romo1 (lower panel), thecells were treated withcycloheximide (CHX, 20g/ml)or trolox. Quantification of theMyc levels following CHXtreatment was carried out bydensitometric scanning in theImageJ program.

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ERK activation and induction of p27Kip1 expression, demonstratingthat ROS originating from the mitochondria play a key role assignaling mediators (Chung et al., 2009; Na et al., 2008). In thepresent study, we also showed that Romo1 knockdown inhibitsERK activation and Myc expression (Fig. 3A), resulting in cellcycle arrest at G1 (Fig. 3C). Therefore, we suggest that the basallevel of ROS derived from the steady-state level of Romo1 isrequired for ERK activation and Myc stabilization. In contrast,excessive ROS produced from increased Romo1 expression inducedby Myc trigger Myc degradation. This suggestion is supported byFig. 3B and Fig. 4F. When the cells were treated with a lowamount of H2O2, Myc expression was increased; however, treatmentwith additional H2O2 down-regulated Myc expression.

Myc is known to play an important role in cell proliferation andits level is controlled transcriptionally, post-transcriptionally orpost-translationally. Abnormal regulation of Myc contributes totumor formation. In the present study, we identified a novel pathwayof negative feedback regulation for Myc during G1 phase. Uponmitogenic stimulation, Myc expression is increased for cell cycleprogression. Myc induces Romo1 expression to enhance cellularROS levels. The ROS promote the cytoplasmic translocation ofSkp2 to cause Myc ubiquitylation, resulting in Myc degradation(Fig. 9). Myc has also been reported to stimulate ROS generation,which induces DNA damage (Vafa et al., 2002). Enhanced ROSlevels were also observed in Myc transgenic animals. We showedthat Myc up-regulation, which was induced by serum stimulation,increased the ROS level via Romo1 expression. Our resultspresented in this study are the first to elucidate the mechanism ofMyc-induced ROS production. An ROS imbalance can causecellular DNA damage and genomic instability, which can contributeto the initiation, promotion and malignancy of tumors (Finkel andHolbrook, 2000). Therefore, the results presented in this studyprovide important information regarding the mechanism of Myc-stimulated oncogenesis associated with ROS.

Materials and MethodsCell culture and reagentsThe human lung fibroblast IMR-90 and WI-38 cells were obtained from the AmericanType Culture Collection (ATCC, Manassas, VA) and cells ranging from 29 to 34 inpopulation doubling level (PDL) were used. WI-38 VA-13 cells were cultured inEagle’s minimal essential media (EMEM, Gibco-Invitrogen, Grand Island, NY).Human embryonic kidney (HEK) 293 cells, HeLa cervix carcinoma cells and Huh-7 human hepatocarcinoma cells were cultured in Dulbecco’s modified Eagle’s media(DMEM, Gibco-Invitrogen). Human non-small cell lung cancer (NSCLC) cell linesA549 and H1299 were cultured in Ham’s F12 and RPMI 1640 medium (Gibco-

Invitrogen), respectively. All media contained 10% heat-inactivated FBS (Gibco-Invitrogen), sodium bicarbonate (2 mg/ml; Sigma-Aldrich, St Louis, MO), penicillin(100 units/ml), and streptomycin (100 g/ml; Gibco-Invitrogen). PD98059, U0126,LY294002, Wortmannin, SP600215 and SB203580 were purchased from StressGen(Victoria, BC, Canada). 3-[[6-(3-amino-phenyl)-1H-pyrrolo[2,3-d] pyrimidin-4-yl]oxy]-phenol (TWS119) was obtained from Calbiochem (La Jolla, CA). N-acetyl-cysteine (NAC), hydrogen peroxide (H2O2), nocodazol,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), stigmatellin,myxothiazol, malonate, rotenone, sodium azide and N-carbobenzoxy-l-leucinyl-l-leucinyl-l-norleucinal (MG132) were purchased from Sigma-Aldrich. 2�,7�-dichlorofluorescein diacetate (DCF-DA) and MitoSOX were obtained from MolecularProbes (Eugene, OR).

PlasmidscDNAs encoding FLAG–Romo1 Wild-type (Wt) and deletion mutants, N and C,were prepared in our laboratory and have been validated previously (Kim et al.,2010). Complementary DNA encoding Myc (human) was cloned into pcDNA3(Invitrogen). pCGN-HA–Myc (Wt) and deletion mutants (A, B, C, D, E, Fand G) were described previously (Herbst et al., 2004; Tworkowski et al., 2002).pCl-FLAG–Myc (Wt) and substitution mutants (T58A, S62A and T58AS62A) andthe pCGN-HA–Fbw7 construct were kindly provided by Keiichi I. Nakayama andMasaki Matsumoto (Department of Molecular and Cellular Biology, KyushuUniversity, Japan) and have been described earlier (Yada et al., 2004). The p3-FLAG–Myc-Fbw7 was kindly provided by Professor Bruce E. Clurman (FredHutchinson Cancer Research Center, University of Washington School of Medicine,Seattle) and was described previously (Welcker et al., 2004). CMV-FLAG–Skp2 waskindly provided by Professor Tae Jun Park (Department of Biochemistry andMolecular Biology, Ajou University, Republic of Korea) and was described previously(Park et al., 2009).

siRNAThe sequences of Romo1 siRNA were unique to their intended targets, based onBLAST searches. The Romo1 siRNA sequence was 5�-GGGCUUC -GUGAUGGGUUG-3� (sense strand). The other siRNA against Romo1 was describedpreviously (Hwang et al., 2007). The Myc siRNA sequences (Grandori et al., 2005),Skp2 siRNA sequences (Carrano et al., 1999; Nishitani et al., 2006; Zhang et al.,2004), and control siRNA sequence (Chung et al., 2009) were described previously.siRNAs were purchased from Bioneer (Taejon, Republic of Korea).

AntibodiesAntibodies were: anti-Myc mouse monoclonal (Santa Cruz Biotechnology, SantaCruz, CA) and rabbit polyclonal (Santa Cruz Biotechnology), anti-p27kip1 mousemonoclonal (BD Pharmingen, San Diego, CA) and rabbit polyclonal (ZymedLaboratories, San Francisco, CA), anti-phospho-ERK rabbit polyclonal (CellSignaling Technology, Beverly, MA), anti-ERK rabbit polyclonal (Cell SignalingTechnology), anti-phospho-Akt rabbit polyclonal (Cell Signaling Technology), anti-Akt rabbit polyclonal (Cell Signaling Technology), anti-Skp2 rabbit polyclonal(Santa Cruz Biotechnology) and anti-Lamin B1 rabbit polyclonal (Santa CruzBiotechnology), -actin mouse monoclonal (Sigma-Aldrich), anti-FLAG (M2)(Sigma-Aldrich) and anti-HA (Sigma-Aldrich). Mouse monoclonal antibody (mAb)against Romo1 was described previously (Kim et al., 2010).

Serum deprivation and stimulationFor serum stimulation experiments, human lung primary fibroblast IMR-90 and WI-38 cells and human embryo kidney (HEK) 293 cells were washed twice with serum-free media and further incubated in EMEM with 0.05% FBS for 48 h (Lee et al.,2010). EMEM containing 30% FBS was then added and cells were collected at theindicated time points.

Semi-quantitative RT-PCR and real-time PCRSemi-quantitative RT-PCR analysis was performed as described previously (Chunget al., 2008). SYBR Green PCR amplifications were performed using an iCycler iQReal-Time Detection System (Bio-Rad Laboratories, USA) associated with theiCycler Optical System Interface software (version 2.3; Bio-Rad). All PCRexperiments were carried out in triplicate with a reaction volume of 25 l, usingiCycler IQ 96-well optical grade PCR plates (Bio-Rad) covered with iCycler optical-quality sealing film (Bio-Rad). Data analyses (calculations), including determiningthe relative amounts of each target mRNA, were performed with the iCycler IQ real-time detection system (Bio-Rad).

Transfection, immunoprecipitation, and western blot analysisCells were transfected with the indicated constructs or siRNA using LipofectamineTM

(Gibco-Invitrogen). The immunoprecipitation and western blot analysis weredescribed previously (Kim et al., 2010).

Measurement of ROS production and immunofluorescence assayIntracellular ROS production was measured using a fluorescence microscope(Olympus LX71 microscope), and the images were analyzed using MetaMorphsoftware (Universal Imaging, Westchester, PA) for quantification purposes as


Fig. 9. A proposed model for Romo1-mediated Myc degradation in anegative-feedback mechanism.

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described earlier (Kim et al., 2010; Lee et al., 2010). For immunofluorescenceassays, cells were fixed in 3.7% paraformaldehyde (Sigma-Aldrich) for 10 minutesat room temperature and stained using standard protocols. For quantification ofprotein translocation, 100–200 cells were monitored in each experiment byfluorescence microscopy and were validated as described previously (Gao et al.,2009; Lin et al., 2009).

Flow cytometric analysisFor analysis of cell cycle profile by FACS, cells were harvested in a time-dependentmanner after induction, fixed with ethanol, stained with propidium iodide (PI, 50g/ml, Sigma-Aldrich) containing RNase A (100 mg/ml, Sigma-Aldrich) for 30minutes at room temperature. The DNA content was analyzed using a FACScan flowcytometer (Becton Dickinson, San Jose, CA).

Cell fractionation assayThe Nuclear extract kit (California, USA) was used to perform cellular fractionationin accordance with the manufacturer’s instructions. The purity of the extract wasconfirmed by western blot analysis against anti-cytosol-specific--actin (Sigma-Aldrich) or anti-nuclear-specific-lamin B1.

Protein stabilization analysis and in vitro ubiquitylation assayFor protein stabilization analysis, HeLa cells were transfected with the indicatedconstructs. After transfection for 48 h, cells were treated with cycloheximide (CHX,20 g/ml). The cell lysates were prepared and analyzed by western blot analysis.After CHX treatment, endogenous or exogenous Myc levels were quantified bydensitometric scanning in the image J program. For Myc ubiquitylation, cells weretransfected with Ubiquitin (Ub)-HA plasmid together with various constructs for 2days and then treated with MG132 (10 M) for 6 h. The immunoprecipitates weresubjected to western blot analysis as described previously (Kim et al., 2010).

Statistical analysisEach assay was performed in triplicate and independently repeated at least threetimes. Statistical significance was defined as P<0.05. Means, SEs and Ps werecalculated using GraphPad PRISM version 4.02 for Windows (GraphPad Software,San Diego, CA).

This research was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF) funded bythe Ministry of Education, Science and Technology (2010-0021371),by a grant from the National R&D Program for Cancer Control,Ministry for Health, Welfare and Family Affairs, Republic of Korea(1020180), by National Nuclear R&D Program through the NationalResearch Foundation of Korea (NRF) funded by the Ministry ofEducation, Science and Technology (20100018574) and by a grant ofthe Korea Healthcare Technology R&D Project, Ministry for Health,Welfare & Family Affairs, Republic of Korea (A084537-0902-0000100).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/11/1911/DC1

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romo1 is a negative-feedback regulator of myc · post-translational level through regulation of its...

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