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Loss of Git2 induces epithelial–mesenchymaltransition by miR146a-Cnot6L-controlled expressionof Zeb1

Wu Zhou1 and Jean Paul Thiery1,2,3,*1Institute of Molecular and Cell Biology, A*STAR, Singapore 1386732Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 1175973Cancer Science Institute, National University of Singapore, Singapore 117599

*Author for correspondence (jpthiery@imcb.a-star.edu.sg)

Accepted 11 March 2013Journal of Cell Science 126, 2740–2746� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126367

SummaryEpithelial–mesenchymal transition (EMT) can be induced by several pleiotropically activated transcription factors, including the zinc-

finger E-box-binding protein Zeb1. Mechanisms regulating Zeb1 expression have been partly uncovered, showing a critical role for themiR-200 family members. In the present study, we show that Zeb1 is regulated by the Arf GTPase-activating protein (GAP) Git2.Following the loss of Git2, we found that miR-146a maturation is enhanced, which in turn promotes the expression of Zeb1 andinduction of EMT. Furthermore, we found that Cnot6L, a validated target of miR-146a, affects the stability of Zeb1 mRNA through its

deadenylase activity. Our results present evidence for a new role for loss of Git2 in promoting EMT through a novel regulatory pathway.

Key words: Git2, miR-146a, EMT, Zeb1

IntroductionEpithelial–mesenchymal transition (EMT) (Thiery and Sleeman,

2006; Thiery et al., 2009; Sleeman and Thiery, 2011) is an

evolutionarily conserved process during morphogenesis that has

more recently been implicated as a driving force promoting the

local invasion and distant dissemination of carcinoma (Thiery,

2002; Kalluri and Weinberg, 2009). Git2 is a member of the GIT

protein family characterized by an N-terminal GTPase-activating

protein domain (Sabe et al., 2006). Git2 show GTPase-activating

activity toward Arf1 and Arf6, which influences membrane

traffic and actin remodelling (Randazzo et al., 2007). Git2

interacts with many molecules, such as G protein-coupled

receptor kinases, PIX [p21-activated kinase (PAK)-interacting

exchange factor] (Bagrodia et al., 1999) and paxillin (Turner

et al., 1999), and serve a pivotal role in focal adhesion

disassembly (Hoefen and Berk, 2006), cell polarity and the

directional motility of migrating adherent cells (Frank et al.,

2006) and neutrophils (Mazaki et al., 2006). Although Git2 is

crucial in the regulation of the migration, adhesion and polarity

of nonhematopoietic cells, part of the function of Git2 may stem

from their ability to repress Rac activation. For example, Git2

represses Rac-dependent lamellipodia extension and cell

spreading in HeLa human cervical cancer cells (Frank et al.,

2006) and in migrating adherent cells (Nishiya et al., 2005).

Because migration, adhesion and polarity are fundamental for

EMT, it is possible that the Git2 has a role in governing EMT.

Here we report loss of Git2 induces EMT by promoting the

expression of Zeb1. We found that miR-146a maturation was

regulated by Git2. Moreover, Cnot6L, a validated target of miR-

146a, affects the stability of Zeb1 mRNA through its deadenylase

activity.

ResultsGit2 is required to maintain the epithelial state inepithelial cells

After screening of a small library of siRNA that could potentially

interfere with EMT of the bladder carcinoma cell line NBT-II,

Git2 was interestingly identified because epithelial morphology

of NBT-II was lost in Git2 siRNA cells (data not shown). To

validate this observation, NBT-II cells were infected with a

lentivirus coding a short hairpin RNA (shRNA) targeting Git2 to

obtain stable clones. Knockdown efficiency tests, as determined

by western blotting, identified clone #2 as the most effective, and

it was subsequently used in this study (Fig. 1A). To test whether

Git2 shRNA was specific to knock down Git2, exogenous Git2

expression was enforced into Git2 knockdown (Git2 KD) cells.

The western blotting results demonstrated that Git2 expression

was complemented by exogenous Git2 (Fig. 1B). NBT-II cells

form epithelial-like compact colonies, with well-defined cell

contacts. However, individual cells in small colonies exhibit

lamellipodial activity at the free edge, inducing rotation of the

colony (Fig. 1C; supplementary material Movie 1). Transduced

NBT-II cells, with lentiviral constructs expressing shRNA

targeting Git2, exhibited an elongated, mesenchymal-like

morphology with modest motility and transient contact with

neighbouring cells (Fig. 1C; supplementary material Movie 2).

Wound healing assay (Fig. 1D) and transwell (Boyden chamber)

cell migration assay (supplementary material Fig. S2B) showed

that silencing of Git2 promoted the migration ability of NBT-II in

vitro. Moreover, the epithelial morphology was rescued when

Git2 was re-expressed into Git2 KD cells (Fig. 1C). Git2 is thus

essential for the maintenance of the epithelial state of NBT-II

cells. We next sought to test whether Git2 played a similar role in

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other epithelial cell lines. Two carcinoma epithelial cell lines,HepG2 and T47D, and one normal epithelial cell line, EpH4 were

selected to test the role of Git2. As anticipated, Git2 knockdownresulted in the loss of compaction of the epithelial colonies withthe most pronounced change in morphology in EpH4 cells

(Fig. 1E).

The miR-146a is augmented in Git2 KD cells

We next performed expression profiling of control and Git2 KDNBT-II cells to identify potential key regulators induced by Git2KD. Overall, 33 genes were downregulated and 15 genes were

upregulated in Git2 KD cells (supplementary material Table S1).Some of the identified genes, such as Cdh1 (E-cadherin), Vim

(vimentin), MMPs and Zeb1 are landmark genes in EMT (Thieryand Sleeman, 2006; Gregory et al., 2008). To confirm themicroarray results, the mRNA and protein levels of Cdh1, Vimand Zeb1 were further assessed by real-time quantitative PCR

and western blotting. The transcriptional and protein levels ofCdh1 were significantly reduced in Git2 KD cells, whereasVim and Zeb1 were, remarkably, increased (Fig. 2A).

Immunostaining of E-cadherin showed that the expression andlocation of E-cadherin were affected by depletion of Git2.Exogenous expression of Git2 in Git2 KD cells reversed the Git2

expression and location to control status (supplementary materialFig. S3A). Together, these results indicated that the loss of Git2repressed epithelial gene expression and promoted EMT.

Zeb1 is a well-established inducer of EMT (Gregory et al.,

2008), and here we show that Git2 inhibition increased Zeb1expression. Meanwhile, the expression of other EMT-relatedtranscriptional factors Snail2 and Twist1 was tested in control,

ShRNA control and Git2 KD cells. Silencing of Git2 didn’tchange the protein level of Snail2 or Twist1 (supplementarymaterial Fig. S2A). As such, we hypothesized that Git2 lies

upstream of Zeb1 in EMT. We speculated that the regulation ofZeb1 by Git2 might be controlled by microRNAs, since somemiRNAs have been identified as regulators of Zeb1 expression

during EMT (Gregory et al., 2008; Brabletz and Brabletz, 2010;Reshmi et al., 2011). Expression profiling of microRNAsrevealed the dramatic augmentation of miR-146a in Git2 KDcells (supplementary material Table S2; Fig. 2B), which was

further confirmed with real-time quantitative PCR (Fig. 2C). InGit2 KD cells, we noticed an increase in only the mature form ofmiR-146a, while pre-miRNA-146a transcripts remained stable

(supplementary material Table S2 microRNA microarray). Wethus suspected that Git2 affects the maturation of miR-146a. Thisnotion was confirmed via real-time PCR quantification of

precursor (pre-mir-146a) and mature miR-146a (miR-146a)transcripts in Git2 KD cells (Fig. 2D, ‘G’, green bars).Moreover, this increase in mature miR-146a transcripts wasreduced when Git2 KD cells were forced to express exogenous

Git2 (Fig. 2D, ‘G+Exo’, black bars). In NBT-II control cells, themature miR-146a was downregulated by almost 50% whenoverexpressing Git2 (Fig. 2E, ‘OE’). However, neither the

enforced Git2 expression in Git2 KD cells nor theoverexpression Git2 in control cells influenced the RNA levelof pre-miR-146a (Fig. 2D,E). Thus, only the maturation of miR-

146a was regulated by Git2.

Arf-GAP activity of Git2 is indispensable for the maturationof miR-146a

Git2 is an Arf GTPase-activating protein (GAP) belonging to theRas superfamily of small GTPases. Arf proteins cycle betweentheir active-GTP-bound and inactive-GDP-bound conformations.

Hydrolysis of bound GTP is mediated by GAP (D’Souza-Schoreyand Chavrier, 2006). The GAP activity of Arf-GAP ensure Arf-GAP bind to activated Arf (Arf-GTP) and stimulate their GTPase

activity. To evaluate the Arf-GAP activity of Git2 on theinfluence of miR-146a maturation, we firstly compared thedifference of Arf-GTP-bound form between Git2 KD and control

cells. GTP-bound Arf were captured by immobilized specific Arfeffector GGA3 (Golgi-associated, gamma adaptin ear containing,Arf binding protein 3) (Hafner et al., 2006). Enhanced levels of

Fig. 1. Git2 is required to maintain the epithelial state in NBT-II cells.

(A) Lentiviral particles expressing Git2 shRNA (constructs #1–5) were used

to infect NBT-II cells. A stable knockdown Git2 cell line was selected with

2.5 mg/ml puromycin. Git2 protein levels were measured by western blotting.

Beta-actin was used as a control. (B) The exogenous Git2 expression was

enforced into Git2 knockdown (Git2 KD) cells. The protein level of Git2 was

measured by western blotting (C, control; SC, shRNA control; G, Git2

shRNA; G+Exo, Git2 shRNA+exogenous Git2). (C) The morphology was

changed in Git2 knockdown (Git2 KD) cells (3), which could be rescued with

forced Git2 expression in Git2 KD cells (4). Scale bars: 15 mm. (D) ShRNA

control cells and Git2 shRNA-transfected NBT-II cells were seeded into a 24-

well tissue culture plate and used for a scratch wound healing assay. Images

were taken immediately after scratching (T0) and 24 hours after scratching

(T24). The silencing of Git2 promoted the migration ability of NBT-II in

vitro. (E) Three other cell lines were infected with lentiviral shRNA particles.

Loss of Git2 resulted in the acquisition of a mesenchymal-like phenotype.

Scale bars: 15 mm.

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GTP-bound Arf proteins were precipitated in NBT-II Git2 KD

cells as compared with control and shRNA control cells (Fig. 3A,

middle panel), suggesting that depletion of Git2 failed tostimulate GTPase activity of Arf and then favours the Arf-

GTP-bound form. Next, we employed wild-type (WT) Git2 and

GAP-inactive C11A mutant Git2 (Cukierman et al., 1995) into

Git2 KD NBT-II control cells. Enforced expression of the C11A

mutant, in which the critical cysteine residue for the GAP

activity, cysteine 11, was mutated to alanine to diminish the GAP

activity, unsuccessfully inhibited the mature miR-146a (Fig. 3B).

In addition, silencing of Arf6 rescued the effect of Git2 KD in

NBT-II cells. The proceeding of EMT and the synthesis of miR-146a were reduced when introducing Arf6 siRNA in Git2 KD

cells (supplementary material Fig. S1A,B). These indicate that

Arf-GAP activity of Git2 is indispensable for the maturation of

miR-146a. To further investigate the role of miR-146a, we

blocked miR-146a with a miR-146a antagomir in Git2 KD NBT-

II cells. The inhibitory efficiency was determined by real-time

PCR (supplementary material Fig. S4A). The miR-146a

antagomir successfully reverted Git2 KD NBT-II cells to an

epithelial morphology (Fig. 3C), re-suppressed the protein levelof Zeb1 and Vim and rescued the expression of Cdh1 (Fig. 3D).

This suggests miR-146a may act as a significant promoter of

EMT, in contrast to the suppressive role of miR200 family and

miR205.

Cnot6L controls the stability of Zeb1

MicroRNAs are small, non-coding RNAs that modulate gene

expression by targeting specific mRNA (Gregory et al., 2008). Toidentify the direct targets of miR-146a, we searched for the

predicted targets using the Targetscan database (http://www.

targetscan.org). We found that putative target genes, Robo1,Cds1 and Cnot6L, were also present in the downregulated gene

list (supplementary material Table S1). Following forcedexpression of Robo1, Cnot6L and Cds1 separately in Git2 KD

NBT-II cells (supplementary material Fig. S3B), we revealed that

only overexpressed Cnot6L could reduce Zeb1 protein levels inGit2 KD cells (Fig. 4A). To determine whether Cnot6L was the

direct target of miR-146a, the PCR products containing mutationof miR-146a seed recognition sequences in the 39-UTR Cnot6L

(Fig. 4C) and WT 39-UTR Cnot6L were inserted into the

luciferase reporter vector. The relative luciferase activity of 39-UTR Cnot6L was reduced in Git2 KD cells. However, the effect

was abolished by mutating the putative miR-146a binding siteswithin the 39-UTR of Cnot6L (Fig. 4B). Moreover, the inhibitory

effect was removed when introducing the inhibitor (antagomir for

miR-146a) to block miR-146a (Fig. 4B). The miR-146a antag-omir failed to change Git2 protein levels, but rescued Cnot6L

expression and repressed Zeb1 expression (Fig. 4D). In addition,NBT-II cells with enforced miR-146a expression inhibited the

expression of Cnot6L, Cdh1 and induced the expression of Vim

Fig. 2. miR-146a is augmented in Git2 KD cells. (A) The

mRNA and protein levels of Zeb1, Vim and Cdh1 were

measured in Git2-depleted and control NBT-II cells. All

experiments were repeated three times. (B) The miRNA

Affymetrix microarray revealed that miR-146a was dramatically

augmented in Git2 KD cells. (C) Quantitative real-time PCR

showed that miR-146a, rather than miR-200 family members,

were significantly increased in Git2 KD cells. All experiments

were repeated three times. (D) Exogenous Git2 was forced to

express in Git2 KD cells (G+Exo). The relative RNAs level of

precursor miR-146a and maturation miR-146a were measured.

All experiments were repeated three times. (E) The maturation

miR-146a was downregulated when overexpressing Git2 in

NBT-II control cells. All experiments were repeated three times.

SC, shRNA control; G, Git2 shRNA; G+Exo, Git2

shRNA+exogenous Git2.

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and Zeb1 (Fig. 4E). These data demonstrated that Cnot6L was a

direct target of miR-146a, and that Git2 regulates Zeb1 through

Cnot6L.

Cnot6L, the catalytic subunit of the Ccr4b–Not complex, is a

key enzyme ensuring deadenylation-dependent degradation of

specific mRNAs (Morita et al., 2007). Northern blot analysis was

carried out to test whether the deadenylase activity of Cnot6L

influenced the stability of Zeb1 mRNA. In Git2-depleted cells,

Zeb1 mRNA was prominently increased, as before. In agreement

with the protein levels in Fig. 4A, forced expression of Cnot6L

decreased Zeb1 mRNA to control levels (Fig. 5A). The

deadenylation and decay of Zeb1 mRNA were monitored by

Northern blotting analysis after transiently transfected with a

gene encoding Cnot6L in Git2 KD NBT-II cells. The amounts of

Zeb1 mRNA isolated at time intervals after transient transfection

showed a gradient reduction (Fig. 5B). To confirm that Cnot6L

affected the stability of Zeb1 mRNA, we next examined the half-

life of Zeb1 mRNA. NBT-II cells were treated with actinomycin

D to inhibit de novo transcription to determine the Zeb1 mRNA

levels at various time points after treatment (supplementary

material Fig. S4B). We found that the rate of decline in Zeb1

transcript levels was lower in Git2-depleted cells than in control

cells, and that re-expression of Cnot6L accelerated the rate of

decline (Fig. 5C). All together, the loss of Git2 seems to induce

the maturation of miR-146a. Increased amounts of the mature

form of miR-146a reduce the levels of Cnot6L, thus preventing

Zeb1 mRNA destabilization. This newly uncovered pathway

results in the execution of the EMT program (Fig. 5E).

DiscussionIn this report, we are unexpected to find that Arf-GAP protein

Git2 is involved into EMT through miR-146a mediated Zeb1

pathway. In contrast to the suppressive role of miR200 family

and miR205, mature miR-146a seems to induce EMT. Our data

demonstrate that Arf-GAP activity of Git2 is necessary for the

maturation of miR-146a. Many proteins have been shown to

regulate the maturation of miRNAs, such as SMAD (Davis et al.,

2008) and small GTPase, Ran (Lund et al., 2004). The classic

Fig. 3. Arf-GAP activity of Git2 is indispensable for the maturation of

miR-146a. (A) The immobilized GST-GGA3-PBD beads were used to

capture GTP-bound ARF. Much more ARF-GTP proteins were pulled down

in NBT-II Git2 KD cells than in control cells. (B) Git2 WT and GAP-inactive

C11A mutant were transfected into Git2 KD cells. Relative miR-146a levels

were detected. C11A mutant was incapable of reversing the RNA level of

miR-146a. (C) The miR-146a antagomir could rescue the altered cell

phenotype (2) to an epithelial cell phenotype (3) in cells treated with

Git2 shRNA. Scale bars: 15 mm. (D) The inhibitory effect of miR-146a on

Cnot6L was abrogated when applying an antagomir to miR-146a. Expression

of Vim, Zeb1 and Cnot6L reverted to control levels. (C, control; SC, shRNA

control; G, Git2 shRNA; G+antagomir, Git2 shRNA+miR-146a antagomir;

WT, wild type).

Fig. 4. Cnot6L controls the stability of Zeb1. (A) Robo1, Cds1 and

Cnot6L cDNAs were transfected into Git2 KD cells. The overexpression

(OE) of Cnot6L inhibited the expression of Zeb1. V, vector. (B) PCR

products containing mutation of miR-146a seed recognition sequences in

the 39-UTR Cnot6L (white bars) and wild type 39-UTR Cnot6L (black

bars) were inserted into the luciferase reporter vector. The relative

luciferase activity of wild type 39-UTR Cnot6L was reduced in Git2 KD

cells. The inhibitory effect was abolished by mutating the putative miR-

146a binding sites within the 39-UTR of Cnot6L. An inhibitor (antagomir

for miR-146a) was introduced into Git2 KD cells to block miR-146a

(G+146a). The miR-146a antagomir reversed relative luciferase activity

of wild type 39-UTR Cnot6L. All experiments were repeated three times.

(C) A scheme to show the construct of wild type and mutant 39-UTR

Cnot6L luciferase reporter system. (D) An antagomir to miR-146a was

applied to block miR-146a (G+146a). The inhibitory effect of miR-146a

on Cnot6L was abrogated. Expression of Zeb1 and Cnot6L were reverted

to control levels. (E) NBT-II cells were transiently transfected with

pcDNA3 vector (V) or expression plasmids for miR-146a (miR-146a).

The expression of Git2, Cnot6L and EMT markers were detected by

western blotting.

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mechanism to regulate the maturation of miRNA includes

nuclear Drosha (Lee et al., 2003) and cytoplasmic Dicer (Lee

et al., 2002). Our evidence points to the Git2-regulated

maturation of miR-146a. However, how Git2 regulate the

maturation of miR-146a? Is it mediated by Drosha or Dicer or

other proteins? These questions were required to be further

investigated.

Epithelial cells are converted into mesenchymal cells by EMT,

a mechanism hypothesized to play a key role in cancer invasion

and metastasis (Thiery and Sleeman, 2006). Traditionally, EMT

can be induced by various growth factors and their downstream

molecules. However, the three different growth factors tested

here successfully increased the expression of Zeb1, but failed to

decrease Git2 expression (Fig. 5D). Moreover, Loss of Git2

didn’t change the RNA level of miR-200 family and miR-205

(Fig. 2C; supplementary material Fig. S3C). This suggests that

there are additional transduction pathways for the fine-tuned

control of Zeb1-mediated EMT. One argument against the role of

EMT in cancer progression is that metastases exhibit phenotypes

similar to their corresponding primary tumours (Tarin et al., 2005;

Christiansen and Rajasekaran, 2006). However, there is increasingevidence to indicate that EMT is not irreversible (Guo et al., 2012;Ocana et al., 2012). The reverse process – mesenchymal to

epithelial transition (MET) – is vital for the completion ofmorphogenesis and during the final stages of differentiation for thetissue anlage (Lim and Thiery, 2011; Sleeman and Thiery, 2011).However, how EMT cells switch back to an epithelial phenotype

through MET is unclear. It is noteworthy that most carcinomaexhibit intermediate phenotypes that exhibit epithelial cellplasticity that is more likely to be responsible for the transiting

between EMT and MET. The newly identified miR-146a mediatedZeb1 pathway, compensating for miR-200 mediated Zeb1pathway, provides the possibility of flexible transition between

EMT and MET. Besides, given that Arf-GAP protein Git2 isproved to be involved in EMT, we propose a model that Arfproteins, cycling between their active-GTP-bound and inactive-

GDP-bound conformations, connect the balance of GAP and GEFto the switch between EMT and MET (Fig. 5F).

Materials and MethodsCell lines and antibodies

NBT-II, HepG2, T47D and EpH4 cells (ATCC, Manassas, VA, USA) were maintainedin complete DME (10% FBS) with antibiotics at 37 C and with 5% CO2. Antibodies andreagents were obtained from the following sources: anti-Git2 (#SAB4503703) and anti-Cnot6L (#HPA042688) antibodies (Sigma–Aldrich, St. Louis, MO, USA); anti-Zeb1antibody (#SC-10572; Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-Vimantibody (550513; BD PharmingenTM, San Diego, CA, USA); anti-Cdh1 antibody(#610182; BD Biosciences, Franklin Lakes, NJ, USA); anti-beta actin antibody(#ab8227; Abcam, Cambridge, MA, USA); anti-Snail2 antibody (#WH0006591M5;Sigma–Aldrich, St. Louis, MO, USA); anti-Twist1 antibody (#LS-C30601-100;LifeSpan BioSciences Company). As secondary antibodies, HRP-conjugated donkey-anti goat (Sigma, Poole, UK), goat-anti rabbit or goat-anti mouse antibodies (GEHealthcare, Chalfont St. Giles, UK) were used. Detection was performed usingWestpico Chemiluminescence substrate (Thermo Scientific).

Lentiviral shRNA

Git2 shRNA (#SC-40637-V) and shRNA control lentiviral particles (#SC-108080;Santa Cruz Biotechnology Inc.) (10 ml) were mixed with 1 ml 10 mg/ml polybrene(#H9268; Sigma–Aldrich) and diluted into 1 ml Dulbecco’s Modified EagleMedium (DMEM) without foetal bovine serum (FBS). The diluted medium withlentiviral particles were added to cells seeded into 24-well plates. 24–48 hourslater, the medium was replaced by DMEM with 10% FBS and 1% penicillin–streptomycin followed by positive selection using 2.5 mg/ml puromycin(#A11138-02; Invitrogen, Carlsbad, CA, USA).

Wound healing assay

NBT-II cells and Git2 KD NBT-II cells were seeded into 24-well tissue cultureplate in a density ,70–80% confluence as a monolayer. Gently and slowly scratchthe monolayer with a new 1 ml pipette tip across the center of the well. Afterscratching (T0), gently wash the well twice with medium to remove the detachedcells. Replenish the well with fresh medium. Grow cells for additional 24 hours(T24). Wash the cells twice with 16PBS. Take photos on a microscope. Eachexperimental group were repeated three times.

Transwell cell migration assay

Control, shRNA control and Git2 shRNA cells were incubated in upper layer ofBoyden chamber coated with matrigel for 24 hours. After incubation, cells onmembrane were washed with PBS three times. Images were taken undermicroscope for 20 times magnification. All experiments were performed understerile conditions.

Live cell imaging

Git2 KD and control NBT-II cells were seeded in 12-well plates. Live cell imagingwas acquired by Zeiss Axiovert 200M Cell Imaging System (Zeiss Microimaging,Thornwood, NY), with a 206objective, and exposure times of 40 ms and 5-minuteintervals.

cDNA and plasmid transfection

cDNAs were obtained from Origene (Rockville, MD, USA): Git2 cDNA(#RG214925), Cnot6l cDNA (#RG219766), Robo1 cDNA (#SC109740), and

Fig. 5. (A) Cnot6L cDNAs were forced to express in Git2 KD cells

(G+Cnot6L). Zeb1 RNA levels were analysed by northern blotting. 28S was

used as a control. Zeb1 mRNA was increased in Git2 KD cells. Forced

expression of Cnot6L decreased Zeb1 mRNA to control levels. (B) Git2 KD

cells were transiently transfected with a gene encoding Cnot6L. The amounts

of Zeb1 mRNA were monitored by northern blotting analysis at time intervals

after transient transfection as indicated. (C) Cells were treated with

actinomycin D to inhibit de novo transcription. Zeb1 mRNA levels were

determined by real time PCR at various time points after treatment. The rate

of decline in Zeb1 transcript levels was lower in Git2-depleted cells than in

control cells. Re-expression of Cnot6L (G+Cnot6L) accelerated the rate of

decline. (D) Three different growth factors (HGF, hepatocyte growth factor;

PDGF, platelet-derived growth factor; TGFb, transforming growth factor-b)

were used to induce EMT. Git2 and Zeb1 protein expression were measured

after 24 hours of stimulation. Ctr, control. (E,F) Schemes to describe the

mechanism of Git2 regulating EMT (E) and a possible connection between

the EMT/MET switch and the Arf-GDP/GTP switch (F).

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Cds1 cDNA (#SC119345). cDNAs (2 mg) were mixed with 3 ml FuGENER 6Transfection Reagent (#E2691; Promega, Madison, WI, USA) and incubated for15 minutes at room temperature. The mixture was added to NBT-II or Git2 KDNBT-II as indicated. The transfected cells were selected in the presence of 600 mg/ml G418 (Invitrogen, #10131-035), and resistant clones were further confirmed bywestern blotting.

The miR-146a antagomir construct (#RmiR-AN0196 am02; GeneCopoeia,Rockville, MD, USA) was transfected into Git2 KD NBT-II cells as describedabove. The transfected cells were harvested and washed in PBS containing 0.2%BSA. Cells (16105 cells per sample) were sorted on a FACSAria II (BDBiosciences) cell sorter equipped with a 594-nm laser.

NBT-II cells were transiently transfected with pcDNA3 vector or expressionplasmids for miR-146a (Addgene plasmid 15902). 48 hours later, cells were lysedby RIPA lysis buffer. Whole proteins were collected and applied for westernblotting.

Git2 KD cells were transfected with 2 Arf6 siRNA (#SI00268100 #SI03054947;Qiagen). 48 hours later, cells were lysed by RIPA lysis buffer. Whole proteinswere collected and applied for western blotting.

Quantitative real-time PCR

For Cdh1, Vim and Zeb1Total RNAs were extracted by Trizol (#15596-026; Invitrogen) andcomplementary DNA (cDNA) was synthesized by reverse transcription kit(#205310; Qiagen). Real-time PCR was subsequently performed in triplicatewith a 1:4 dilution of cDNA using the SYBRH Green RT-PCR Reagents Kit. Thecomparative Ct method was used to compute relative expression values. AllmRNA quantification data were normalized to GAPDH.

For mature miR-146a and pre-mir-146aTotal miRNA was extracted using miRNA Iso Kit (#AM1560; AppliedBiosystems, Foster City, CA, USA), according to the manufacturer’sinstructions. MiRNAs were reversed transcribed with TaqmanH microRNA RTKit (#4366596; Applied Biosystems). Real-time PCR was subsequently performedin triplicate using Taqman kits for mature miR-146a (#4427975), pre-mir-146a(#4427012) and miR-200a (#4378069; Applied Biosystems). Data were collectedand analysed using the Rotor-gene software. Fold changes of miR-146a and miR-200a were normalized to control cells. The miR-146a and pre-mir-146aquantification data were normalized to miR-200a.

Microarray analysis

For Git2 microarray analysisTotal RNA samples from Git2 KD and control NBT-II cells were isolated using theRNA isolation kit (#74104; Qiagen) according to the manufacturer’s protocol.Total RNA (100 ng) was reverse transcribed to produce cDNA/mRNA hybridmolecule, which was subsequently used as a template to create double strandedcDNA with a unique DNA/RNA heteroduplex at one end. The cDNA was thenamplified via SPIA (Single Primer Isothermal Amplification), which producessingle stranded anti-sense DNA. Post-SPIA modification generates sense targetcDNA that was fragmented, biotin labeled and hybridized to Affymetrix Rat Gene1.0 ST arrays for 18 hours at 45 C rotated at 60 rpm (Affymetrix, Santa Clara, CA,USA). Arrays were then washed and stained using the FS450_0007 fluidicsprotocol and scanned using an Affymetrix 3000 7G scanner.

For miRNA microarray analysisTotal RNA samples from Git2 KD and control NBT-II cells were isolated by usingthe miRNeasy Mini Kit (#217004; Qiagen) according to the manufacturer’sprotocol. Genisphere FlashTag Biotin HSR labeling techniques was utilized tohybridize samples to Affymetrix miRNA 2.0 Arrays. The miRNA 2.0 Arrayprovides probe sets for 131 species including rat, and comprises more than 15,000unique probe sets for maturation miRNA based on Sanger miRBase (v.15), over2,000 pre-miRNA sequences (Affymetrix). Mature miRNAs are assigned the‘rno_’ prefix and pre-miRNAs ‘hp_ rno_’ as displayed in supplementary materialTable S2.

39-UTR luciferase reporter analysis

The Cnot6l luciferase reporters were purchased from Applied Biological Materials,Inc. (Richmond, BC, USA). The mutant construct with a mutation of the miR-146aseed sequence was generated with the mutagenic oligonucleotide primers (F: 59-AUACUUUCUAGAACUCAGAAUAA R: 59-AUGAGAGAAAAUUUGGGCA-UU), according to the manual of GeneTailor Site-Directed Mutagenesis System(Invitrogen). Cells were co-transfected with each reporter construct and the Renillaluciferase vector pRL-TK (#E2241; Promega), and incubated with passive lysisbuffer, according to the dual-luciferase assay manual. The luciferase activity wasmeasured with a luminometer (Lumat LB9507, Berthold Tech., Bad Wildbad,Germany). The firefly luciferase signal was normalized to the Renilla luciferasesignal for each individual analysis. All experiments were performed in triplicatewith data pooled from at least two independent experiments.

Northern blot analyses

Total RNAs were isolated with ISOGEN according to the manufacturer’s protocol(Nippon Gene, Tokyo, Japan). Northern blot analyses were carried out as describedpreviously (Yoshida et al., 2000). To prepare the probes, DNA fragments of Zeb1 and28S were amplified by PCR. The probes were labelled with [a-32P] dCTP by a random-prime labelling system and were hybridized at 65 C for 2 hours in ExpressHybHybridization Solution (Clontech Laboratories, Inc. Mountain View, CA, USA).

Assays of mRNA stability

Git2 KD cells were transiently transfected with Cnot6L cDNA plasmids, and totalRNA was extracted at the indicated time points and analysed by Northern blotting. Thelevel of Zeb1mRNA was monitored by Northern blotting. 28S was used as control.

Half-life assay

ShRNA control, Git2 KD and Cnot6L overexpressed Git2 KD cells were treatedwith actinomycin D (2.5 mg/ml). Total RNAs were extracted by Trizol (#15596-026; Invitrogen) and complementary DNA (cDNA) was synthesized by reversetranscription Kit (#205310; Qiagen). Real-time PCR was subsequently performedat various time points after actinomycin D treatment in triplicate with a 1:4 dilutionof cDNA using the SYBRH Green RT-PCR Reagents Kit.

Fluorescent staining of E-cadherin

ShRNA control, Git2 KD and Git2 exogenous Git2 KD cells were 100% methanolfixed (5 minutes) and then incubated in 1%BSA/10% normal goat serum/0.3 Mglycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein–protein interactions. The cells were then incubated with the E-cadherin antibody (#ab11512) at 5 mg/ml overnight at +4 C. The secondary antibody(green) was Alexa FluorH 488 goat anti-rat IgG (H+L) used at a 1/1000 dilution for1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 mM.

Pull down assay

Arf6-GTP pull down assay was carried out according to the manual (#16122;Pierce Biotechnology, Rockford, IL, USA). Briefly, Git2 KD cells, shRNA controlcells and control NBT-II cells were washed with PBS at 4 C, harvested into 500 mllysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1% TritonX-100, 0.1% SDS, 0.5% deoxycholate, 5% glycerol, 1 mM phenylmethylsulfonylfluoride, 9 nM pepstatin, 9 nM antipain, 10 nM leupeptin, and 10 nMchymostatin). Cell extracts were incubated with 30 mg of GST-GGA3 or GSTimmobilized on glutathione-Sepharose for 1 hour at 4 C. The pellets were washedthree times with lysis buffer. Bound proteins were eluted by 30 ml elution buffer.The reactions were analysed by immunoblotting with an ARF6-specificmonoclonal antibody.

Western blotting

Total cell lysates (40 mg of protein) were separated by sodium dodecyl sulfate-PAGE (SDS-PAGE) in 8% gels and electrotransferred on to nitrocellulosemembranes. After blocking with 5% skim milk, the membranes were probed withprimary antibodies, respectively, at 4 C overnight (dilution, 1:1000). Themembrane was incubated with appropriate peroxidase-labelled secondaryantibodies and developed by Super Signal chemiluminescence substrate(#34077; Pierce Biotechnology). Beta-actin protein levels were used as a controlfor adequacy of equal protein loading.

Statistics

All data were expressed as mean6s.e.m. Statistical analysis was performed usingthe Student’s t-test as indicated.

AcknowledgementsWe thank Dr Chu Yeh-shiu for support with microscopy; Dr JormayLim for help with Targetscan; Dr Jing Ma and Sim Wen Jing for helpwith cell culture. We thank Prof. Hisataka Sabe for the C11A mutantplasmid. The authors confirm that they have no competing financialinterests.

Author contributionsZ.W. designed and performed the experiments. J.P.T. wrote thearticle. Both authors read and approved the final manuscript.

FundingThis work was supported by A*STAR core funding.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126367/-/DC1

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