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RESEARCH ARTICLE

STAT3 and MCL-1 associate to cause a mesenchymalepithelial transition

A. P. Renjini, Shiny Titus, Prashanth Narayan, Megha Murali, Rajesh Kumar Jha and Malini Laloraya*

ABSTRACT

Embryo implantation is effected by a myriad of signaling cascades

acting on the embryo–endometrium axis. Here we show, by using

MALDI TOF analysis, far-western analysis and colocalization and

co-transfection studies, that STAT3 and MCL-1 are interacting

partners during embryo implantation. We show in vitro that the

interaction between the two endogenous proteins is strongly

regulated by estrogen and progesterone. Implantation, pregnancy

and embryogenesis are distinct from any other process in the

body, with extensive, but controlled, proliferation, cell migration,

apoptosis, cell invasion and differentiation. Cellular plasticity is

vital during the early stages of development for morphogenesis

and organ homeostasis, effecting the epithelial to mesenchymal

transition (EMT) and, the reverse process, mesenchymal to

epithelial transition (MET). STAT3 functionally associates with

MCL-1 in the mammalian breast cancer cell line MCF7 that

overexpresses STAT3 and MCL-1, which leads to an increased

rate of apoptosis and decreased cellular invasion, disrupting the

EMT. Association of MCL-1 with STAT3 modulates the normal, anti-

apoptotic, activity of MCL-1, resulting in pro-apoptotic effects.

Studying the impact of the association of STAT3 with MCL-1 on

MET could lead to an enhanced understanding of pregnancy and

infertility, and also metastatic tumors.

KEY WORDS: STAT3–MCL-1 interaction, Apoptosis, Invasion,

Epithelial mesenchymal transition, MET

INTRODUCTIONSuccessful implantation, and thereby pregnancy, is a hormone-

driven process. Several essential cytokines and growth factors

regulate the steroidal actions of hormones in order to prepare

the endometrium for implantation. STAT3, a versatile member

of the family known as ‘signal transducers and activators of

transcription’ (STAT), mediates the axial responses of cytokines.

STAT3 is involved in normal cellular responses, as well as

oncogenesis (Takeda and Akira, 2000). In resting cells, STATs,

including STAT3, are localized to the cytoplasm. Upon activation

by cytokines, STATs are known to be phosphorylated by Janus

kinases, which leads to formation of homo- or heterodimers

through interactions between the Src homology 2 (SH2) domains

and phosphorylated tyrosine residues. STAT3 then rapidly

translocates to the nucleus, where it increases the expression of

target genes. STAT3 plays an essential role in interleukin (IL)-9-induced expression of primary response genes, such as c-Mycand Cited2 (Zhu et al., 1997). Increased phosphorylation of STAT3

is known to be associated with elevated expression of potentialdownstream targets of STAT3 – these include the genes encodingapoptosis inhibitors [survivin, MCL-1, HSP27 (also known asHSPB1), adrenomedullin and Bcl-xL], cell cycle regulators (cyclin

D1, cyclin-dependent kinase inhibitor 1, c-Fos, MAP2K5 and c-Myc) and inducers of tumor angiogenesis [vascular endothelialgrowth factor, COX-2 (also known as PTGS2) and the matrix

metalloproteases (MMP)-2, MMP-10 and MMP-1] in invasivebreast cancer tissues (Hsieh et al., 2005; Sinibaldi et al., 2001). Thepleiotropic response of STAT3 has been attributed to its ability

to act downstream of receptors for a number of IL-6-familycytokines, including IL-6, IL-11, ciliary neuorotrophic factor,oncostatin M, leukemia inhibitory factor (LIF) and interferonfamily members (IL-10, IFN-c and IFN-a) (Heinrich et al., 1998;

Kisseleva et al., 2002). Furthermore, STAT3 has been shownto mediate transcriptional responses to granulocyte colony-stimulating factor, leptin (Akira, 1997), receptor tyrosine kinases

(epidermal growth factor, colony stimulating factor 1 and platelet-derived growth factor) and IL-2 family members (IL-2, IL-7 andIL-15) (Zhong et al., 1994).

Members of the IL-6 family of cytokines are expressed duringimplantation in the mouse (Bhatt et al., 1991) and human

(Charnock-Jones et al., 1994; Nachtigall et al., 1996). In vivo

STAT3 activation, induced by LIF alone, is restricted to day 4 ofpregnancy, resulting in the localization of STAT3 specifically to

the nuclei of cells in the luminal epithelium, which coincides withthe onset of uterine receptivity (Cheng Jr et al., 2001). Targeteddisruption of Stat3 revealed that this protein is essential for earlyembryonic development (Takeda et al., 1997). STAT3 signaling

also plays a crucial role in the induction of antigen-specific T-celltolerance (Cheng et al., 2003). Thus, in order to elucidate the roleof STAT3 in the process of embryo implantation, we investigated

which proteins this transcription factor interacts with at the timeof embryo implantation.

In this study, we show that STAT3 physically and functionallyinteracts with its downstream target myeloid cell leukemia-1(MCL-1) during the ‘window of implantation’. We demonstrate the

interaction using co-immunoprecipitation, far-western analysis,colocalization of the two proteins in uterine sections, and with co-transfection studies in MCF7 cells, and show that the interactionis strongly mediated by the ovarian hormones estrogen and

progesterone. The epithelial mesenchymal transition (EMT) ischaracterized by changes in protein expression, increased invasionand resistance to apoptosis. In this study, we show that STAT3

interacts with MCL-1, which leads to increased apoptosis,decreased invasion and upregulation of certain proteins, such asE-cadherin (also known as cadherin-1) and cytokeratins, which are

normally downregulated during the EMT.

Utero-Embryo Repromics Lab, Division of Molecular Reproduction, Rajiv GandhiCentre for Biotechnology, Thycaud PO, Poojappura, Thiruvananthapuram 695014, Kerala, India.

*Author for correspondence (laloraya@rgcb.res.in; laloraya@gmail.com)

Received 10 July 2013; Accepted 1 January 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1738–1750 doi:10.1242/jcs.138214

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RESULTSMALDI-TOF analysis of STAT3-immunoprecipitated sampleLike many proteins, STAT3 mediates its action by interactingwith a number of other proteins. Some of the known interactingpartners of STAT3 are coactivators, such as CBP–p300 andNCoA/SRC1a, which possess intrinsic histone acteyltransferase

activity and enhance the transcriptional activity of STAT3(Giraud et al., 2002). TIP60 (also known as KAT5) interactswith STAT3; it binds to the a-chain of the IL-9 receptor,

suggesting that Tip60 might be involved in IL-9 signaling (Xiaoet al., 2003). Although STAT3 is known to play a key role duringembryo implantation, the protein networks that it interacts with in

the uterus are not known. Thus, we initiated experiments toidentify partners of STAT3 that enable it to perform its functions.

Immunoprecipitation of STAT3 using nuclear or cytosolic

extracts, prepared from uteri, resulted in a limited number ofprotein bands that stained positively with Coomassie dye. Eachband was trypsin digested and then subjected to matrix-assistedlaser desorption/ionization (MALDI) analysis. The results

identified possible interacting partner proteins of STAT3(Fig. 1A,B). The band at the 37-kDa-position in the nuclearimmunoprecipitation (GB8) was subjected to MALDI analysis,

this analysis strongly pointed towards the possibility of theprotein being MCL-1, a member of the BCL-2 family proteins,which regulate apoptosis (Fig. 1C). MCL-1 has sequence

similarity to BCL-2. BCL-2 is involved in normal lymphoiddevelopment and a t(14; 18) chromosome translocation in thegene results in lymphoma (Kozopas et al., 1993). The presence of

STAT3 in the nuclear immunoprecipitate was confirmed bypeptide fingerprint, followed by Mascot analysis, of the band at88 kDa (GB4) (Fig. 1C). Therefore, we focused our efforts

towards establishing the relationship between the STAT3 andMCL-1 proteins during the time of embryo implantation.

Coimmunoprecipitation of STAT3 and MCL-1 implicatesMCL-1 as an interacting partner of STAT3 at the windowof implantationTo establish the authenticity of the interaction between STAT3and MCL-1, we investigated whether MCL-1 co-precipitatedwith STAT3. Using antibodies against STAT3, STAT3 was

immunoprecipitated from uterine nuclear and cytosolic extracts.An MCL-1-positive band was detected when the STAT3precipitates were probed with antibody to MCL-1 (Fig. 1D,E).

The presence of STAT3 in these immunoprecipitates wasconfirmed by western blotting with an antibody against STAT3.For nuclear and cytosolic extracts, protein input was shown by

histone H3 and actin, respectively (lower panels). We furtherconfirmed the interaction by immunoprecipitating MCL-1 fromthe nuclear (Fig. 2A) and cytosolic (Fig. 2B) extracts fromdifferent days of pregnancy. MCL-1 immunoprecipitates showed

a STAT3-positive band (Fig. 2Ca) in the uterine nuclear extractson all the tested days of pregnancy, suggesting the existence of anassociation between STAT3 and MCL-1 during the window of

implantation. The western blot of MCL-1 confirms the presenceof MCL-1 in the immunoprecipitate (Fig. 2Cb). In the case ofimmunoprecipitation using the uterine cytosolic extracts, MCL-1

did not pull down STAT3, and vice versa, in the cytosol from thesample taken on day 4 (D4) 10:00 (Fig. 2Cb). A possibleexplanation is that the low MCL-1 levels indicate a lack of

cytosolic distribution at D4 10:00 and thus might be unavailablefor interaction with STAT3 at this stage. It is evident from theresults of the immunoprecipitations that the STAT3–MCL-1

Fig. 1. Immunoprecipitation of STAT3 from uterinecytosolic and nuclear extracts on different days ofpregnancy. (A,B) STAT3 was immunoprecipitatedfrom (A) nuclear and (B) cytosolic extracts taken ondifferent days of pregnancy: D4 10:00, D4 16:00, D505:00 and D5 10:00. Co-precipitated proteins wereseparated by SDS-PAGE and stained usingCoomassie dye. (C) Peptide mass fingerprinting dataof proteins interacting with STAT3 were analyzed usingMASCOT software. GB represents the gel bandnumber. The protein score is the sum of the highestions score for each distinct sequence and indicates thelevel of significance. The score is -10*LOG10(P),where P is absolute probability. (D,E) Western blot ofSTAT3 (upper panel) and MCL-1 (middle panel) onSTAT3 immunoprecipitates taken on different days ofpregnancy using nuclear (D) and cytosolic (E) extracts.The lower panels show the protein input, representedby actin in the cytosolic fraction and Histone H3 innuclear fraction. M denotes protein marker.

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interaction is prominent in the nucleus at the early peri- and peri-implantation period (D4 16:00 and D5 05:00), which is then

reduced in the post-implantation period (D5 10:00). Although theSTAT3–MCL-1 interaction is retained in the cytosol during theperi-implantation phase, there is a conspicuous lack of interaction

between the two proteins in the cytosol at the pre-implantationperiod (D4 10:00). Mock-immunoprecipitation and secondary-antibody-alone controls show the specificity of the antibodiesused (supplementary material Fig. S1).

Far-western analysis confirms the STAT3–MCL-1 interactionTo test the interaction between STAT3 and MCL-1, far-western

analysis was performed. The day D4 16:00 cytosolic extract,where the interaction between STAT3 and MCL-1 wasprominent, was used as the prey. Purified STAT3 protein was

used as the bait and bovine serum albumin (BSA) was used as acontrol. The blot probed with an antibody against STAT3

revealed the classical 41 kDa band that indicates MCL-1L, anMCL-1 variant that is encoded by the myeloid cell leukemia-1

gene and known to migrate at 40–42 kDa. The band indicatingMCL-1L is marked with an asterisk and arrow (Fig. 2Da). Theposition of the band was confirmed when the blot was developed

using an antibody against MCL-1 (Fig. 2Db). The secondary-antibody alone shows two nonspecific albumin-immunopositivebands (NS1 and 2) in the cytosolic extract and BSA samples(Fig. 2Dc). Fig. 2Dd shows the protein load by Coomassie

staining. The membrane that was probed with an antibody againstSTAT3 showed several bands because recombinant STAT3would have also interacted with other proteins in the cytosolic

extract.

STAT3 and MCL-1 interact in vitroTo establish further an interaction between STAT3 and MCL-1, weperformed co-immunoprecipitation with an MCL-1 antibody using

Fig. 2. STAT3 interacts with MCL-1.(A,B) MCL-1 was immunoprecipitated fromnuclear (A) or cytosolic (B) extracts takenon different days of pregnancy: D4 10:00,D4 16:00, D5 05:00 and D5 10:00. Thearrowheads and asterisks indicate STAT3and MCL-1, respectively.(C) Western blot for STAT3 and MCL-1 onMCL-1 immunoprecipitates taken ondifferent days of pregnancy using(a) nuclear extracts (b) cytosolic extracts.M denotes protein marker. (D) Far-westernanalysis of the D4 16:00 cytosolic extractwas used as the prey for far-westernblotting. Purified mSTAT3 protein wasused as the bait and BSA was used as acontrol to perform the experiment.(a) The membrane probed with anantibody against STAT3 showed severalbands; the band representing MCL-1 isindicated by the arrow. (b) The position ofthe band is confirmed by the blotdeveloped with MCL-1. (c) The secondaryalone shows the nonspecific binding ofBSA prominent in both blots(d) protein load by Coomassie staining.CE; cytosolic extract, M, marker; NS1 andNS2, nonspecific band 1 and 2,respectively. (E). Western blot ofimmunoprecipitation with an antibodyagainst MCL-1 using total extracts fromcontrol cells (no transfection), cellstransfected with STAT3 alone, MCL-1alone or STAT3 and MCL-1 incombination. The arrows indicate thespecific protein bands. E2, estrogen.

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total extracts from MCF7 cells transfected with STAT3 or MCL-1alone, or with STAT3 and MCL-1 in combination. The MCL-1

immunoprecipitates were then western blotted with an antibodyagainst STAT3, which showed that STAT3 interacts with MCL-1in cells overexpressing STAT3 (Fig. 2E).

Colocalization of STAT3 and MCL-1 in uterine sections duringembryo implantationHaving established that an interaction between MCL-1 and

STAT3 occurs, we sought to identify the expression pattern of thetwo proteins during the crucial time of embryo adhesion in orderto understand the functional relevance of the interaction.

Consequently, immunocytochemical studies were performed tovisualize the localization of STAT3 and MCL-1 in the uterinesections on different days of pregnancy. STAT3 was labeled with

secondary antibody conjugated to fluorescein isothiocyanate(FITC) and MCL-1 was labeled with secondary antibodyconjugated to tetramethylrhodamine (TRITC). The nuclei of theendometrial epithelium is seen to be devoid of MCL-1 and

STAT3 as both proteins show localization to the membrane. Bycontrast, stromal cell nuclei showed weak STAT3 expression atD4 10:00 of pregnancy. An increased expression of STAT3 and

MCL-1 is evident at D4 16:00 in comparison with D4 10:00(Fig. 3A,B) with their localization prominently in the membraneof the endometrial epithelia and cytosol, as well as nuclei of

stromal cells. This increased expression is maintained at peri-implantation stages but there is a spatio-temporal variation in thedistribution pattern. The early peri-implantation uteri (D4 16:00)

and peri-implantation uteri (D5 05:00) are evident by thepresence of STAT3 and MCL-1 in the the nuclei of cells in thestroma and, the stromal expression of the proteins showing a

dotted appearance, suggesting that the two proteins accumulate innuclear speckles (Fig. 3B). The fluorescence, indicating STAT3

and MCL-1, was redistributed as the uterus prepared to receive aninvasive-phase embryo – showing that STAT3 began to translocatefrom the nucleus to membrane and cytosolic compartments instroma. STAT3 also showed increased expression in epithelial cells

at the late peri-implantation time period, D5 10:00 (Fig. 3A). Theremaining STAT3 and MCL-1 continued to colocalize in nuclearspeckles (Fig. 3B). Reorganization of MCL-1 and STAT3 in the

nucleus of cells towards the endometrial epithelium and stroma,where the embryo has to implant, could signal the death of cells inthe luminal epithelium and stroma in order to accommodate the

invading embryo. The overlapping expression pattern of STAT3and MCL-1 further strengthens the possibility that the two proteinsassociate, and their presence in the implantation site indicates that

STAT3 and MCL-1 play a key role in embryo implantation.

Estrogen and progesterone stimulate time-dependentcolocalization of MCL-1 and STAT3 in MCF7 cellsAs implantation is regulated by the steroidal hormones estrogenand progesterone, we investigated the effect of these twohormones on the interaction between MCL-1 and STAT3 in a

time-dependent manner. The experiments were performed usingMCF7 cells, a cell line in which STAT3 and MCL-1 havepreviously been shown to be expressed (Ding et al., 2007;

Berishaj et al., 2007). The results showed that, before treatmentwith hormones (0 hour), STAT3 and MCL-1 were located in thecytosol (Fig. 4A). In agreement with reported nuclear shuttling of

STAT3 (Meyer and Vinkemeier, 2004), STAT3 was alsoobserved in the nucleus. After 30 minutes of treatment withestrogen, STAT3 began to accumulate in foci in the nucleus, and

Fig. 3. STAT3 colocalizes with MCL-1 in vivo.(A) Colocalization of STAT3 and MCL-1 inuterine sections of different days of pregnancy:D4 10:00, D4 16:00, D5 05:00 and D5 10:00.STAT3 was labeled with FITC (green) and MCL-1 was labeled with TRITC (red). Cell nuclei werestained using DAPI (blue). Arrows indicatenuclear speckles. (B) Corresponding zoomedimage of overlay (indicated by the white boxes),which clearly shows the nuclear speckles.Secondary-alone controls were performed usingFITC- and TRITC-conjugated antibodies (SecFITC and Sec TRITC, respectively). LE, luminalepithelium; LU, lumen; S, stroma. Arrowsindicate nuclear speckles. Scale bars: 10 mm.

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reduced levels of STAT3 were observed in the cytosol; however,

the STAT3 remaining in the cytosol was seen to colocalize withMCL-1. After 24 hours of treatment with estrogen, the majorityof STAT3 became nuclear, whereas a substantial portion of

MCL-1 remained cytosolic; however, in the nucleus, STAT3 andMCL-1 colocalized in a dotted pattern (Fig. 4B). Colocalizationof STAT3 with MCL-1 was predominantly in the nucleus after

30 minutes and 24 hours of progesterone treatment (Fig. 4A,B).The pattern of expression was comparable to that after 24 hoursof estrogen treatment. The same pattern of expression was seen

when progesterone and estrogen were given in combination for24 hours. Hence, both hormones regulate the expression andnuclear translocation of STAT3 and MCL-1. The expression ofSTAT3 and MCL-1 is more targeted to peri-nuclear bodies

(indicated by the yellow dots in the merged image of STAT3 andMCL-1 fluorescence, and the white dots in the merged images ofSTAT3, MCL-1 and DAPI fluorescence; Fig. 4A,B). This

pronounced colocalization of STAT3 with MCL-1, which isespecially apparent in nuclear bodies, is still visible after 24 hoursof treatment with either hormone alone, or in combination

(Fig. 4A,B). Treatment of the cells with estrogen andprogesterone together causes a large fraction of MCL1 andSTAT3 to relocate to the cytosol and also results in reducedexpression of the two proteins.

Exogenously expressed STAT3 and MCL-1 interact in MCF7cellsThe MCF7 cells were co-transfected with STAT3–EGFP andMCL-1–DsRed using Lipofectamine 2000 (Invitrogen). Theproteins were expressed for 48 hours after transfection, andthe pattern of expression was observed by using a confocal

microscope. As shown in Fig. 5A, colocalization of STAT3 andMCL-1 was restricted to the cytosol before treatment withhormones. Although some nuclear expression of STAT3 is seen,

the nuclei are devoid of MCL-1 at t50 (0 hour). After 24 hoursof treatment with 1 nM estrogen, both STAT3 and MCL-1showed increased accumulation in the nucleus, a substantial

fraction also surrounded the nucleus. The merged image clearlyshows overlay of staining of the proteins, indicating that theycolocalize in the cell. After 24 hours of treatment with 10 nM

progesterone, a fraction of STAT3 but not MCL-1 entered thenucleus, even though the two proteins clearly colocalized in thecytosol. This mimics the scenario of D4 10:00 and D4 16:00uteri, which are not subjected to 24 hours of estrogen surge in

vivo. The lack of endogenous MCL-1 entry into the nucleus incomparison with the nuclear entry of overexpressed exogenousMCL-1 in MCF7 cells (Fig. 4 compared with Fig. 5A) could

be due to the different expression levels of the proteins.STAT3 and MCL-1 evidently colocalized in the nucleus whenestrogen and progesterone were given in combination (Fig. 5A),

which is in accordance with the colocalization of endogenousSTAT3 and MCL-1 in MCF7 cells. This is also in line with ourobservations in uterine sections of D5 05:00, that is, a stage

equivalent to 24 hours of estrogen treatment in a progesterone-stimulated environment (Fig. 3). Thus, it appears that bothhormones are required for efficient functioning of STAT3 andMCL-1.

The interaction of STAT3 with MCL-1 requires the PID andSH2 domain of STAT3Having demonstrated an interaction between MCL-1 and STAT3,we wanted to probe which domains of STAT3 mediate thisinteraction. MCF7 cells were transfected, as described above,

with MCL-1–DsRed and constructs encoding domains of STAT3tagged with enhanced green fluorescent protein (EGFP), namelythe protein interaction domain (STAT3-PID), alpha domain,DNA-binding domain (DBD) and SH2 domain (STAT3-SH2)

(Fig. 5B). Colocalization of MCL-1 with STAT3-PID andSTAT3-SH2 in the nucleus was detected by 24 hours ofestrogen treatment; however, in transfections using the

constructs encoding the STAT3 alpha domain and DBD, MCL-1 did not enter the nucleus (Fig. 5C), suggesting that MCL-1interacts with PID and SH2 domains to enter the nucleus.

Levels of phosphorylated STAT3 are reduced uponassociation with MCL-1JAK-mediated tyrosine phosphorylation regulates thedimerization of STATs and is a prerequisite for theestablishment of a classical JAK–STAT3 signaling pathway.Tyr705 phosphorylation is attributed to STAT3 activation

(Bowman et al., 2000; Decker and Kovarik, 2000; Levy andLee, 2002); therefore, we examined the phosphorylation status ofSTAT3 upon its interaction with MCL-1. We immunoprecipitated

MCL-1 from MCF7 cells that had been co-transfected withplasmids encoding STAT3 and MCL-1 and treated with orwithout estrogen. The precipitates were then probed for

phosphorylated STAT3. We observed decreased levels of

Fig. 4. Colocalization of STAT3 and MCL-1 is hormone dependent.(A) Colocalization of STAT3 and MCL-1 in MCF7 cells upon treatment withthe hormones estrogen (E; 1 nM) and progesterone (P; 10 nM). Sampleswere stained after 30 minutes or 24 hours of treatment. An untreated samplewas used as control (0 hour). STAT3 was probed with primary antibody andlabelled using a secondary antibody conjugated to Alexa Fluor 488 (green)and MCL-1 was probed with primary and labeled using an Alexa-Fluor-568-conjugated secondary antibody (red). DAPI was used to stain the nucleus(blue). (B) Corresponding zoomed images of the overlay (indicated by thewhite box) clearly showing the nuclear speckles. Secondary-alone controlsfor both Alexa Fluor 488 and Alexa Fluor 568 are shown (Sec 488 and 569,respectively). Arrows indicate nuclear speckles.

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Fig. 5. Co-transfection of STAT3–EGFP and MCL-1–DsRed in MCF7 cells. (A) The cells were imaged by using a Nikon confocal microscope. Cells weretreated with estrogen alone, progesterone alone or estrogen and progesterone in combination for 24 hours. Clear colocalization of STAT3 (green) and MCL-1(red) in the nucleus was observed in cells treated with both estrogen and progesterone. Nuclei were stained using DAPI (blue). (B) Domain structure of STAT3:PID, protein interaction domain; alpha domain; DBD, DNA-binding domain; SH2, Src homology 2 domain. (C) Co-transfection of domains of STAT3 with MCL-1–DsRed in MCF7 cells before and after estrogen treatment. Clear colocalization of STAT3 and MCL-1 in the nucleus was observed in cells transfected with thePID and SH2 domains after 24 hours of estrogen treatment. E or E2, estrogen; P, progesterone; 0 hour, untreated control. Scale bars: 10 mm.

Fig. 6. Functional relevance of the STAT3 interaction with MCL-1. (A) Phosphorylation status of STAT3 in MCF7 cells co-transfected with STAT3 and MCL-1with (+E2) or without estrogen (2E2) treatment. Immunoprecipitation with an antibody against MCL-1 was performed from whole cell extracts of co-transfectedcells, and the immunoprecipitates were probed for MCL-1 and phosphorylated STAT3 (p-STAT3). Western blotting of the whole-cell extracts for actin wasperformed as control for the level of protein input. (B) Reporter assay showing the efficiency of transactivation of STAT3 responsive promoters in the presence orabsence of estrogen and overexpressed MCL-1 (n56). Stat P, STAT3 promoter reporter. (C) Cell cycle analysis of cells transfected with STAT3, MCL-1 or vectoralone (pcDNA) or co-transfected with STAT3 and MCL-1. (D) Apoptosis assay using a Dual Apoptosis Assay Kit measuring the degree of apoptosis uponoverexpression of STAT3 and/or MCL-1 in MCF7 cells. E or E2, estrogen; P, progesterone.

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phosphorylated STAT3 in estrogen-treated cells that had been co-transfected with MCL-1 and STAT3 (Fig. 6A).

The interaction of STAT3 with MCL-1 modulates the activityof STAT3 on promotersTo analyze whether the activity of STAT3 at the promoters of

response genes is affected by the interaction with MCL-1,luciferase assays were performed using the Cignal STAT3Reporter Assay Kit. Plasmids encoding STAT3 and MCL-1 were

transfected into MCF7 cells along with the STAT3-responsiveluciferase construct. The constitutively expressing Renilla

construct encodes the Renilla luciferase reporter gene and acts as

an internal control for normalizing transfection efficiencies andmonitoring of cell viability. Luciferase activities of the test sampleswere normalized with promoter-alone controls and then plotted. In

cells transfected with STAT3 and MCL-1, the assay showed a 4-fold decrease in the activity of STAT3 on the promoter upontreatment with 1 nM estrogen in comparison with cells withoutestrogen (P,0.003) (Fig. 6B). The STAT3 promoter activity was

high in the absence of estrogen in co-transfected cells. Thecontrols, STAT3 alone and MCL-1 alone, showed high STAT3promoter activity in the absence of estrogen. In the presence of

estrogen, cells transfected with STAT3 alone showed a 21-folddownregulation (P,0.0196) of promoter activity. By contrast,cells transfected with MCL-1 alone showed a statistically

insignificant downregulation of promoter activity in estrogen-treated cells compared with untreated cells that had beentransfected with MCL-1 (P,0.05904) (Fig. 6B). As the presence

of MCL-1 rescued the inhibition of the transcriptional potential ofSTAT3 upon treatment with estrogen from 21-fold to 4-fold, theresults indicate that MCL-1 is a positive modulator of STAT3function in the presence of estrogen and acts to relieve the

repressive effects of estrogen on STAT3 promoter activity.

MCL-1 induces G2/M arrest in transfected cellsTo analyze whether the STAT3–MCL-1 interaction varies withthe cell cycle dynamics, we performed cell cycle analysis usingfluorescence-activated cell sorting. The cells were synchronized

using serum starvation and then treated with estrogen. Weobserved a substantial increase in the number of cells in the G2/Mstage when cells were transfected with only MCL-1, or co-transfected with STAT3 and MCL-1 compared with controls

(Fig. 6C). This correlates with earlier reports that transienttransfection with MCL-1 increases the expression of phospho-Ser345 Chk1 in the absence of DNA damage and leading to

accumulation of cells in G2 (Jamil et al., 2008), which suggests anew role for MCL-1 in generating an appropriate response toDNA damage and in the maintenance of chromosome integrity

(Jamil et al., 2008).

Coexpression of STAT3 with MCL-1 modulates apoptosis inresponse to steroid hormonesThe loss of uterine epithelial cells surrounding the embryo iscrucial for bringing the blastocyst into close proximity with theuterine endometrium for successful embryo implantation. It has

been shown previously that the epithelial cells are phagocytosedby trophoblast cells after apoptotic cell death (Parr et al.,1987);hence, apoptosis is vital during embryo implantation, and we,

therefore, used the Dual Apoptosis Assay Kit to measure thedegree of apoptosis stimulated by the association of STAT3 withMCL-1 in MCF7 cells. Cells transfected with MCL-1 alone

showed a 2-fold increase in apoptosis in the presence of estrogen;

by contrast, cells transfected with STAT3 alone showed a 2.7-foldincrease in apoptosis in the presence of estrogen (Fig. 6D). This

correlates with the downregulation of STAT3 promoter activity inthe presence of estrogen. A previous report has shown thatinhibition of STAT3 activity induces apoptosis and promotesexpression of anti-apoptotic genes in pancreatic cancer cell lines

(Glienke, 2011). In cells co-transfected with STAT3 and MCL-1,the degree of apoptosis was reduced to a level that is less than thatof cells transfected with MCL-1 alone (,73% reduction) in the

presence of estrogen. Hence, MCL-1 protects cells from the effectof estrogen on STAT3 activity, proving the anti-apoptotic role ofMCL-1. The action of progesterone on these transfected cells was

similar to that of estrogen on cells transfected with STAT3 orMCL-1 individually. Cells transfected with MCL-1 alone showeda 1.8-fold increase in the levels of apoptosis in the presence of

progesterone, and cells transfected with STAT3 alone showed a2.6-fold increase in the presence of progesterone. However, whenSTAT3 and MCL-1 were coexpressed, the degree of apoptosiswas increased to almost 5-fold in the presence of progesterone,

suggesting that MCL-1 and STAT3 together cannot rescue theapoptotic effect of progesterone. In the presence of both estrogenand progesterone, cells transfected with MCL-1 alone showed a

5-fold increase in apoptosis, STAT3 alone showed an increase of3.5-fold and coexpression of STAT3 and MCL-1 increased theapoptosis rate by 6-fold in comparison with cells that were left

untreated. Thus, it appears that estrogen and progesterone have anadditive effect on apoptosis and that expression of MCL-1 isunable to abrogate this response.

Interaction of STAT3 with MCL-1 perturbs the epithelial tomesenchymal transitionThe EMT is a well-defined phenomenon that is characterized by

the increased expression of certain proteins, such as N-cadherin,vimentin, MMP-2, fibronectin, SNAI1 and SNAI2, and thedecreased expression of E-cadherin, cytokeratin, desmoplakin

and occludin. In MCF7 cells co-transfected with STAT3 andMCL-1, we detected increased expression of E-cadherin andcytokeratin, which was most pronounced in STAT3 and MCL-1

co-transfected cells treated with estrogen and progesterone.Interestingly, a decreased expression of N-cadherin andvimentin was detected in the co-transfected cells (Fig. 7A).Progesterone, which is known to increase expression of vimentin

(Lin et al., 2003; Uchida et al., 2012), has the same effect in co-transfected cells, but estrogen alone or in combination withprogesterone reverses this effect. Thus, it appears that cells co-

transfected with STAT3 and MCL-1 and treated with estrogenand progesterone in combination are equivalent to implantation-state cells because they exhibit a mesenchymal to epithelial

transition (MET).Functional markers of the EMT include increased invasion and

resistance to apoptosis. Coexpression of STAT3 with MCL-1 in

MCF7 cells resulted in increased apoptosis upon treatment withestrogen and progesterone, as shown in Fig. 6B. As a marker forcell invasion, we analyzed the expression of MMP-2 and itsregulator TIMP-2. Although the level of MMP-2 was found to be

increased in cells treated with estrogen and progesterone together,its levels were clearly downregulated in cells treated withprogesterone alone, compared with that of control, and,

concomitantly, TIMP-2 expression was high in cells co-transfected with STAT3 and MCL-1 in all hormone treatments(Fig. 7B). This can be explained by the fact that estrogen and

progesterone modulate the window of implantation but embryo

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invasion occurs at the time when only progesterone is present and

the effects of estrogen have diminished.To confirm the effects of STAT3 and MCL-1 in promoting a

MET, real-time analysis of the EMT markers was performed

using cells transfected with vector (pcDNA), STAT3 or MCL-1alone or STAT3 and MCL-1 together. The relative expressionwas calculated using the DDCt method with 18S rRNA as the

endogenous control. Estrogen-treated untransfected cells servedas the calibrator. The primers used for the real-time experimentsare listed in the supplementary material Table S2. The data

obtained for each marker corroborated with the results obtainedby western blotting. The mesenchymal marker vimentin(P,0.02) showed a significant decrease in expression comparedwith pcDNA-transfected control cells and N-cadherin (P,0.05)

showed a significant decrease in comparison with STAT3-transfected cells (Fig. 7C). By contrast, the epithelial markersE-cadherin (P,0.03) and cytokeratin 7 (P,0.02) showed an

increased expression in estrogen-treated cells that had been co-transfected with STAT3 and MCL-1, which suggests thatinduction of the MET occurs in these cells (Fig. 7C). The

invasion markers MMP-2 (P,0.02) and MMP-9 (P,0.01)exhibited decreased expression in cells co-transfected withSTAT3 and MCL-1 and treated with estrogen; by contrast, theMMP inhibitor TIMP-2 showed a corresponding increase in

expression in co-transfected cells compared with the control(Fig. 7C), which points to a possible decrease in invasivecapabilities of these cells.

Invasion assays confirm the efficacy of the MET in cellsco-transfected with STAT3 and MCL-1As the real-time expression data indicated that co-transfection ofMCF7 cells with STAT3 and MCL-1 might inhibit the invasive

potential of these cells, we performed invasion assays using the BD

BioCoat Tumor Invasion Fluoroblok cell culture insert. The datashowed that, indeed, cells co-transfected with STAT3 and MCL-1had reduced potential to invade the membrane in response to the

chemoattractant FBS. Compared with cells transfected withSTAT3 alone (P,0.001) and MCL-1 (P,0.0002) alone, cellstransfected with both plasmids showed a decreased invasion

capacity (Fig. 7D). Cells transfected with empty pEGFP-N1 andpDsRedExpress-C1 vectors served as controls.

DISCUSSIONThe foremost finding of our work is the demonstration that, usingpeptide mass fingerprinting, MCL-1 is associated with STAT3during embryo implantation. MCL-1, a gene first identified in a

screen for differentiation-induced genes activated in the humanmonocyte leukemia cell line (Kozopas et al., 1993), was isolatedduring the early differentiation of a human embryonic carcinoma

cell line, an event that serves as a model of early embryogenesis(Sano et al., 2000). Expression of MCL-1 is observed when a cellis committed to differentiate, suggesting that it might be a

crucial factor at decision points determining cell fate. Increasedexpression of MCL-1 is often associated with cell survival,whereas decreased expression corresponds to cell death (Craig,2002). Given the capacity of MCL-1 to promote cell survival

and its presence at various junctures of proliferation anddifferentiation, it has been suggested that MCL-1 has a possiblerole in the immune response and reproduction. Previous work has

shown the role of MCL-1 in oocyte survival versus atresia, fertility(affecting ovulation), embryogenesis and placental development(Sano et al., 2000). Thus, our finding that MCL-1, along with

STAT3, is a key protein required for implantation points to anotherpossible mechanism for regulation of STAT3 action.

Fig. 7. STAT3 and MCL-1 interacts to perturb EMT. Markers of EMT were investigated in MCF7 cells transfected with pcDNA (control), STAT3 alone, MCL-1alone or STAT3 and MCL-1 in combination. (A) Western blot of EMT markers in transfected MCF7 cells: N-cadherin, Vimentin, E-cadherin and cytokeratin 7.Actin was used as control. (B) Western blot for the cell invasion marker MMP-2 and its regulator TIMP-2 in transfected MCF7 cells (C) Real-time gene expressionanalysis of the EMT markers in transfected MCF7 cells. The relative expression was calculated using the DDCt method using 18S rRNA as the endogenouscontrol. Estrogen-treated untransfected cells served as the calibrator. Means6s.e.m. are shown (n53) (D) Invasion assay using transfected MCF7 cells. Control,empty EGFP and DsRed vectors; STAT3–EGFP indicated by green; MCL-1–DsRed indicated by red. Representative images are shown. The assay wasquantified using the Infinite 200 Tecan Microplate reader.

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Our results demonstrate that STAT3 can be co-precipitatedwith MCL-1, that the two proteins colocalize during the late pre-

and peri-implantation period in uterine sections and that STAT3can bind to MCL-1 using far-western blotting analyses. Co-transfection studies with various domains of the STAT3 proteins

identified the PID and SH2 domain of STAT3 as being importantfor the interaction with MCL-1. It is known that STAT3 interactswith other proteins using its PID and SH2 domain (Zhang et al.,1999). STAT3-mediated expression of MCL-1 is essential for

the survival of primary human macrophages that have beendifferentiated in vitro (Liu et al., 2003). Studies have revealed aSTAT3 regulatory element in the MCL-1 promoter, and

transfection with dominant-negative STAT3 diminished MCL-1mRNA and protein levels (Isomoto et al., 2005). Chromatinimmunoprecipitation analysis has demonstrated a direct binding

of STAT3 to the putative STAT3-binding sequences in the MCL-1 promoter (Isomoto et al., 2005). A STAT3-binding serum-inducible element (SIE) at position 286 to 293 has beenidentified in the MCL-1 promoter region, and activated STAT3

can bind to the SIE-related element in the murine MCL-1promoter, which indicates a possible role for STAT3 in regulatingexpression of MCL-1 (Epling-Burnette et al., 2001a). Although

STAT3 has been previously shown to bind to the MCL-1promoter, our work here is the first report of a direct interactionbetween the two proteins.

Steroid hormones orchestrate the process of embryoimplantation and pregnancy. As shown by our data, steroidhormones (estrogen and progesterone) modulate the expression of

both MCL-1 and STAT3, their co-translocation into the nucleusand the promoter activity of STAT3. Steroid hormones are knownto modulate STAT3 nuclear translocation and affect itstranscriptional activation potential (Wang et al., 2001). Our

results support these established findings and, additionally, showthat MCL-1 translocates with STAT3 under hormonal control. Thedistinct localization in nuclear bodies, after estrogen and

progesterone treatment, has important functional implications.Nuclear bodies include the well-characterized Cajal bodies,the nucleolus, perinucleolar and perichromatin regions, and

promyelocytic leukemia (PML) nuclear bodies (which have animportant role in DNA replication, surveillance, and repair, as

well as mRNA and rRNA synthesis and assembly). Nuclearbodies are very dynamic and mobile within the nuclear space

and are regulated by cellular stress, such as heat shock,apoptosis, senescence, exposure to heavy metals, viral infectionand DNA damage responses (Zimber et al., 2004). STAT3 has been

reported to be present in these nuclear bodies (Herrmann et al.,2004); thus, the colocalization of STAT3 with MCL-1 in nuclearbodies will have implications in mediating STAT3-directedfunctions.

Our cell cycle analysis, which showed increased G2/M arrest incells transfected with MCL-1 alone or co-transfected with STAT3and MCL-1, is in line with the data of Jamil and colleagues,

which shows that increased levels of MCL-1 induce G2/M arrest(Jamil et al., 2008). This suggests that the association of STAT3with MCL-1 does not affect the cell cycle, in comparison with the

transfection of MCL-1 alone.Apoptosis plays an important regulatory role in mammalian

embryogenesis and development. MCL-1 is an anti-apoptoticBCL-2-related gene, MCL-1 deficiency results in peri-implantation

embryonic lethality due to an inability of the embryo to implant in

utero, and the null blastocysts fail to develop or attach in vitro,indicating a defect in the trophectoderm; however, the inner cell

mass could grow in culture. This indicates that MCL-1 is essentialfor preimplantation development and implantation, suggesting that ithas a function beyond regulating apoptosis (Rinkenberger et al.,

2000). Cooperative regulation of MCL-1 by Janus kinase, STAT andphosphatidylinositol 3-kinase contributes to delayed apoptosis inhuman neutrophils, which is stimulated by granulocyte-macrophage

colony-stimulating factor (Epling-Burnette et al., 2001b). MCL-1 isan important anti-apoptotic factor for the survival of T cells atmultiple stages of their life-cycle in vivo. MCL-1 is essential for thesurvival of double-negative and single-positive thymocytes, as well

as naive and activated T cells. MCL-1 functions together with Bcl-xL to promote double-positive thymocyte survival (Dzhagalov et al.,2008). MCL-1 plays an important role in the development of various

carcinomas (Shigemasa et al., 2002; Zhou et al., 2001). The invadingperi-implantation embryo has been, rightly, compared to invasivetumor cells; yet, a notable difference is that the embryo is

differentiated into the trophectoderm and the inner cell mass. Theoverall expression of MCL-1 can increase during the induction of cell

Fig. 8. The involvement of STAT3 and MCL-1 inthe EMT–MET shift. The function of STAT3 andMCL-1 alone and in combination is depicted. STAT3and MCL-1, individually, promote the EMT due toincreased N-cadherin, vimentin and MMPs. Ourfindings prove that STAT3 and MCL-1 combine topromote the MET, and the reversal of EMT ischaracterized by increased E-cadherin andcytokeratin, and decreased N-cadherin, vimentin andMMP.

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death, as well as during the induction of differentiation (Zhan et al.,1997). The role of steroid hormones, together with the interaction

between STAT3 and MCL-1, increase the understanding of theprocess of apoptosis of uterine cells during embryo implantation.

Our apoptosis assay using MCF7 cells demonstrates thatsteroid hormones, especially progesterone in combination with

estrogen, can increase the rate of apoptosis in cells transfectedwith both STAT3 and MCL-1, and that the STAT3–MCL-1interaction changes the function of MCL-1 from an anti-apoptotic

to a pro-apoptotic protein, which correlates with the reportthat progesterone inhibits growth and induces apoptosis inbreast cancer cells through downregulation of BCL-2 and the

upregulation p53 (Formby and Wiley, 1999). Our luciferase assaydata show that estrogen downregulates the activity of STAT3 onpromoters, which was almost restored by MCL-1, lending further

support to our hypothesis.The passage of a cell through the EMT is marked by several

discrete processes, such as activation of transcription factors,cytoskeletal remodeling and differential expression of surface

proteins and ECM components. Previous reports suggest thatspheroids formed from the choriocarcinoma cell line JAR, underthe influence of estrogen and progesterone, induce the EMT in

Ishikawa cells (Uchida et al., 2012). Our analysis of the EMTmarkers found in cells overexpressing STAT3 and MCL-1demonstrate that the association of STAT3 with MCL-1 prevents

the EMT. We found increased expression of E-cadherin andcytokeratin and decreased expression of N-cadherin, vimentinand MMP-2. The western blot data of the EMT markers was

further confirmed with real-time gene expression analysis andinvasion assays. Real-time analysis showed a substantialincrease in epithelial markers and subsequent downregulationof mesenchymal markers. Invasion assays and real-time analysis

demonstrated that cells co-transfected with STAT3 and MCL-1have less invasive potential. In addition, our observation of asubstantial reduction in phosphorylated STAT3 in estrogen-

treated cells that had been co-transfected with STAT3 and MCL-1 enabled us to hypothesize that lower levels of phosphorylatedSTAT3 could have implications for the EMT-to-MET shift.

Thus, the STAT3–MCL-1 interaction controls the switch froman EMT phenotype to the MET phenotype (Fig. 8). Our resultsare strongly supported by the recent observation that METoccurs during decidualization when fibroblast-like endometrial

stromal cells are converted into polygonal epithelium (Zhanget al., 2013). The colocalization of STAT3 with MCL-1 isclearly evident in stromal cells at the post-implantation stage,

which is in line with the onset of decidualization. Increasedapoptosis and decreased invasion contribute to the disruptionof EMT by the interaction of STAT3 with MCL-1. We

hypothesize that overexpressed MCL-1 suppresses STAT3activity by interacting with it, serving as a control mechanismfor successful embryo implantation. This interaction serves as a

control to prevent excessive embryo invasion, in contrast totumors, which are characterized by rapid and extensive growthand invasion in the host.

In summary, our results lead us to postulate that STAT3

interacts with MCL-1 in the uterus during embryo implantation.MCL-1 is known to control two distinct processes, apoptosisand the cell cycle; our demonstration of an interaction with

STAT3 now suggests that MCL-1 might have other, currentlyunknown, functions as a result of its association with STAT3.Our studies suggest that the association of MCL-1 with STAT3

causes a MET and is, thus, crucial for regulating epithelial

plasticity during implantation. Further work will be essential toinvestigate the role of the interaction between MCL-1 and

STAT3 during implantation failure, development and theprogression of cancer.

MATERIALS AND METHODSAnimalsSwiss strain albino mice, with a regimen of 14 hours of day and 10 hours

of night, were fed with water and food ad libitum. Swiss strain male mice

of proven fertility were used for mating the females. The day of the vaginal

plug was designated as day 1 of pregnancy. Uterine tissue was extracted

from the females, cleared of adhering fat and retrograde flushing with PBS

was performed to remove the embryos. Embryos were observed by a

microscope to determine the correct stage of pregnancy. All animal work

was approved by the institute’s Animal Ethical Committee.

Cytosolic and nuclear extract preparationTissues, taken from different days of pregnancy, were thawed on ice and

washed with PBS (10 mM). The tissues were minced and homogenized in

buffer [10 mM PIPES, 100 mM KCl, 1 mM MgCl2, 1.5 mM EGTA,

3 mM NaCl, 3.4 mg PMSF, phosphatase inhibitor cocktail, complete mini

protease inhibitor cocktail (Roche)] using the Polytron homogenizer. The

homogenate was then incubated on a Genei Rocker-100 on ice for

30 minutes and centrifuged at 200 g at 4 C for 5 minutes in an Eppendorf

5820R centrifuge to pellet unbroken cells. The supernatant was aspirated

and centrifuged at 1000 g at 4 C for 10 minutes to separate the nuclei. The

post-nuclear supernatants were further centrifuged at 12,000 g at 4 C to

remove mitochondria and other large organelles. The obtained supernatant

was centrifuged at 100,000 g at 4 C in a Beckman L8-M ultracentrifuge for

1 hour to separate membrane extract. The supernatant obtained (cytosolic

extract) was stored at 280 C. The nuclear pellets obtained after centrifuging

at 1000 g were washed with PBS (three times, 10 minutes each) and

vigorously pipetted up and down. The nuclear protein fraction was extracted

on a rocking bench for 30 minutes in hypertonic buffer [20 mM HEPES,

10 mM KCl, 1 mM MgCl2, 400 mM NaCl, 1 mM EDTA, 1 mM

dithiothreitol (DTT), 1 mM sodium orthovanadate, 200 mM PMSF, 20%

glycerol, 1% Triton X-100, phosphatase inhibitor cocktail, complete mini

protease inhibitor cocktail (Roche)]. The homogenate was then subjected to

centrifugation at 20,000 g at 4 C for 30 minutes. The supernatant collected

(nuclear extract) was stored at 280 C. Protein concentrations were

determined using the Bio-Rad DC Protein assay kit.

Immunoprecipitation and MALDI TOF analysis to identifyinteracting partnersFor the identification of the interacting partners, immunoprecipitation

studies followed by MALDI TOF analysis were performed as described

previously (Padmanabhan et al., 2011). Briefly, 200 mg of nuclear or

cytosolic extracts were incubated with 1 mg of primary antibody. The

extracts were then incubated with protein A agarose at room temperature

for 60 minutes and then centrifuged in an Eppendorf 5820R centrifuge

(Eppendorf, Hamburg, Germany) at 14,000 r.p.m. at 4 C for 30 minutes.

The pellet was denatured in Laemmli buffer and the proteins were

separated electrophoretically by SDS-PAGE (Laemmli, 1970). The gels

were stained using Coomassie and the protein bands were subjected to

trypsin digestion using a Trypsin Profile IGD Kit (Sigma) as per the

manufacturer’s protocol. The peptide fragments were then subjected to

analysis and the results computed using Mascot (http://www.

matrixscience.com/). Mock immunoprecipitation was also performed to

prove the specificity of the antibodies (supplementary material Fig. S1).

SDS-PAGE and western blottingImmunoprecipitated samples were separated by SDS-PAGE and

transferred to a polyvinylidene fluoride (PVDF) membrane as

described previously (Towbin et al., 1979). The blots were developed

using the following primary antibodies: STAT3 (sc482), MCL-1 (sc819),

vimentin (sc5565), E-Cadherin (sc7870), N-cadherin (sc31031), MMP-2

(sc13595), TIMP-2 (sc5539), cytokeratin 7 (sc17116), p-STAT3

(sc7993). All primary antibodies were purchased from Santa Cruz

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Biotechnology. Secondary labeling was performed using suitable

secondary antibodies. To visualize the band, the membrane was

either subjected to color development using 3,39-diaminobenzidine

tetrahydrochloride dihydrate (DAB) as an electron donor, H2O2 as the

enzyme substrate and NiCl2 as metal enhancer, or was visualized using

an enhanced chemiluminescence detection system. Images were captured

using a Bio-Rad FluorS Multi Imager. The specificity of the antibodies

was monitored by performing secondary-antibody-only controls

(supplementary material Fig. S1).

Far-western analysisThe far-western analysis technique was employed to identify proteins

that interacted, as described previously (Kihm et al., 1998). Samples

[cytosolic extract (D4 16:00, CE), BSA and purified STAT3]

were resolved using SDS-PAGE on a 10% gel and were then

electrophoretically transferred to a PVDF membrane. The PVDF

membranes were incubated in a blocking solution (50 mM Tris-HCl,

50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3% BSA, pH 7.9) for 1 hour at

4 C. Recombinant STAT3 protein (200 ng in 10 ml blocking solution)

was added and the membrane was gently agitated for 2 hours at 4 C.

The membrane was thoroughly washed with washing buffer (20 mM

Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2% Tween-20,

pH 7.9) three times for 10 minutes each, and bound protein was detected

by immunoblotting with STAT3. As a control, another panel of samples

was probed with an antibody against MCL-1. A control using secondary

antibody alone was also performed.

Colocalization studies in uterine sectionsThe endogenous peroxidase activity was quenched using 3% hydrogen

peroxide in absolute methanol. Antigen unmasking was performed by

autoclaving slides for 10 minutes after placing them in a coplin jar with

10 mM sodium citrate buffer (pH 6.0). After cooling, the slides were rinsed

in PBS and then incubated with blocking solution (5% normal goat serum)

for 1 hour. The washed slides were incubated with the primary antibody

(1:200 dilution) overnight at 4 C. After extensive washing with PBS, the

sections were incubated with the fluorescein isothiocyanate (FITC)-

conjugated secondary antibody in the dark for 60 minutes. The unbound

secondary antibody was washed off by repeated buffer replacements. The

slides were then washed and blocked and stained for the second protein,

using a tetramethylrhodamine (TRITC)-conjugated secondary antibody, as

described above. To detect the nuclei, the sections were further processed for

staining by incubation in 1:1000 dilution of 50 mg/ml solution of DAPI for

10 minutes. After proper washing, the sections were again dehydrated and

then imaged using a Leica TCS SP2 confocal laser scanning microscope. All

experiments were repeated five times. Secondary-antibody-alone controls

were performed for both FITC- and TRITC-conjugated antibodies.

Colocalization of endogenous MCL-1 and STAT3 upon treatmentwith estrogenTo identify the localization of the proteins under in vitro conditions,

studies were performed using the MCF7 cell line. The cells were

maintained in Dulbecco’s modified Eagle’s medium (DMEM) with

nonessential amino acids, 1% antibiotic-antimycotic and 10% fetal

bovine serum. The cells were grown to 80% confluence in the culture

flask. 0.25% trypsin-EDTA was then used to detach and isolate the cells.

Cells were seeded on to coverslips at a density of 105 cells per well in a

24-well culture plate. The cells were supplemented with their respective

media and allowed to grow. Water-soluble estrogen and progesterone

were administered to cells at concentrations of 1 nM and 10 nM per well,

respectively. The coverslips were taken out at 0, 30 minutes or 24 hours

after the treatment with estrogen or progesterone, or a combination

of the two, after 24 hours. The cells were then fixed using 4%

paraformaldehyde, neutralized in 50 mM ammonium chloride and

permeabilized with 0.25% Triton X-100. The cells were blocked with

5% goat serum, probed with the primary antibody (STAT3, catalogue

number SC-482, Santa Cruz Biotechnology; MCL-1, catalogue number

SC-819, Santa Cruz Biotechnology, 1:200 dilution) and fluorochrome-

conjugated secondary antibody (1:200 dilution). DAPI was used to

stain the nucleus. After thorough washings, the coverslips were

mounted on the slides using 1:1 glycerol:PBS. Images were captured

using a Nikon confocal microscope. Secondary-antibody-alone controls

were performed for both Alexa-488- and Alexa-568-conjugated

antibodies.

Construction of vectorsTotal RNA was prepared from mouse uterus using Trizol (Sigma). The

purity of the total RNA was established by determining the A260/280 ratio

and the concentration was then adjusted (100 ng–5 mg/ml). cDNA from

total RNA was prepared using Ready-To-Go T-primed First-Strand

Synthesis kit (GE Healthcare), which utilizes the moloney murine

leukemia virus (M-MuLV) reverse transcriptase and an oligo(dT)18

primer. All oligonucleotides were purchased from Ocimum Biosolutions.

Stat3 and Mcl-1 were amplified from mouse uterine cDNA, and the

sequence showed complete similarity with the available Genbank

sequences of Stat3 transcript variant 1 (accession no. NM_213659) and

Mcl-1 (accession no. NM_008562.3), respectively. The product of

Stat3 was cloned into pEGFP-N1 (Clontech) for co-transfection

studies and pcDNA 3.1(-) (Invitrogen) for luciferase and apoptosis

assays. The primers used to generate STAT3–EGFP were: STAT3_

forward XhoI 59-GGTGGTCTCGAGATGGCTCAGTGGAACCAGC-

TG-39 and STAT3_reverse EcoRI 59-GGTGGTGAATTCGCATGGG-

GGAGGTAGCACACT-39, and for STAT3 pcDNA STAT3_pcDNA_

forward 59-CTCGAGGTTATGGCTCAGTGGAACCAGCTG-39 and

STAT3_pcDNA_reverse 59-GAATTCTCACATGGGGGAGGTAGC-

ACACT-39 were used. Mcl-1 was cloned into pDsRedExpress-C1

(Clontech) using primers MCL-1_forward EcoRI 59-GAATTCTATGTT-

TGGCCTGCGGAGAAACGCGGT-39 and MCL-1_reverse SalI 59-

GTCGACCTATCTTATTAGATATGCCAGACCAGC-39. MCL-1 pcDNA

was generated using primers MCL-1_pcDNA_forward 59-GAATT-

CACCATGGTTGGCCTGCGGAGAAAC-39 and MCL-1_pcDNA_

reverse 59-GGATCCCTATCTTATTAGATATGCCAGACC-39. EGFP-

bound constructs of different domains of STAT3 [protein interaction

domain (PID), alpha domain, DNA-binding domain (DBD) and SH2

domain] were also generated for co-transfection assays with MCL-1–

DsRed (supplementary material Table S1).

Co-transfection assays with STAT3 and MCL-1 to establish theinteraction between the proteinsThe MCF7 cell line was cultured in DMEM media (Gibco-BRL)

supplemented with 10% FBS (Sigma) and appropriate antibiotics.

For transfection, the cells were incubated with Opti-MEM (Life

Technologies) without antibiotics for 20 minutes. The cells were then

transfected with plasmids encoding STAT3–EGFP and MCL-1–DsRed by

using the Lipofectamine reagent (Invitrogen). The appropriate amount of

plasmids and the Lipofectamine reagent were diluted separately in Opti-

MEM and incubated at room temperature for 5 minutes (0.8 mg plasmid

per 50 ml of Opti-MEM; 1 ml Lipofectamine per 50 ml Opti-MEM). The

two mixtures were then combined and incubated for 20 minutes at room

temperature.100 ml of the mix was added to each well and incubated in 5%

CO2 for 4–6 hours. Fresh medium, supplemented with FBS and antibiotics,

was added after 6 hours. We also performed co-transfection assays of

MCL-1–DsRed with EGFP constructs of different domains of STAT3:

protein interaction domain (PID), alpha domain, DNA-binding domain

(DBD) and SH2 domain. MCF7 cells transfected with empty EGFP and

empty DsRed plasmids served as negative controls.

Reporter assayTo analyze whether the activity of STAT3 on promoters is affected by a

STAT3–MCL-1 interaction, luciferase assays were performed using a

Cignal STAT3 reporter assay kit from SABiosciences. The cells (106 per

well) were seeded in a 12-well tissue culture plate the day before

transfection. Cells were transfected with constructs encoding STAT3 and

MCL-1, along with a STAT3-responsive luciferase construct, which

contains the firefly luciferase reporter gene under the control of a

minimal cytomegalovirus promoter and tandem repeats of the SIE (sis

inducible element) transcriptional response element. Each sample was

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tested in triplicate. After transfection, the cells were incubated with 1 nM

of estrogen and then lysed using the passive lysis buffer (Dual Luciferase

Assay kit, Promega) 24 hours after transfection. Luciferase assays were

performed with 20 ml of the cell extract and 100 ml of luciferase assay

buffer. The enzyme activity was measured for 2 seconds using a TD 20/

20 luminometer.

Apoptosis assayApoptosis is a key event during the process of embryo implantation. We

used a Dual Apoptosis Assay Kit with NucView2488 Caspase-3 Substrate

and Sulforhodamine 101-Annexin V kit (Biotium) for measuring the

degree of apoptosis created by the association of STAT3 with MCL-1

using MCF7 cells. The Dual Apoptosis Assay Kit allows simultaneous

detection of caspase-3 activation and phosphatidylserine translocation in

apoptotic cells. The assay was performed as per the manufacturer’s

instructions using the Tali2 Image-based cytometer (Invitrogen). Cells

were transfected with STAT3 pcDNA, MCL-1 pcDNA or STAT3 and

MCL-1 in combination. Hormone treatments [estrogen (1 nM) or

progesterone (10 nM) individually, or estrogen and progesterone in

combination] were performed to analyze the effect of hormones on

apoptosis induced by the association of STAT3 with MCL-1.

Microscope image acquisitionColocalization of STAT3 and MCL-1 in uterine sections was imaged

using a Leica TCS SP2 Confocal Laser Scanning Microscope DMIRE2

using a663 NA 1.4 objective lens. Images were captured using a Leica

camera (DC350F) and the Leica acquisition software. Co-transfection

studies and endogenous localization of MCL-1 and STAT3 in MCF7

cells were observed using a Nikon Confocal Microscope with the

objective lens Plan Apo VC 606 oil DIC N2, NA 1.4. Images were

captured using the Nikon acquisition software.

Cell culture experimentsMCF7 cells were cultured in 35-mm dishes and cells were transfected

with vector alone, STAT3 or MCL-1 alone or STAT3 with MCL-1 in

combination. The cells were treated with 1 nM of estrogen for

24 hours after transfection. These cells were used for flow-cytometric

analysis of cell cycle progression and relative quantification of EMT

markers.

Cell cycle analysisFor analyzing the cell cycle status, after transfection and treatment with

estrogen, cells were fixed with 70% ethanol at 4 C overnight and

subsequently stained with propidium iodide solution [0.1% (v/v) Triton

X-100, 10 mg/ml propidium iodide (Sigma) and 100 mg/ml DNase-free

RNase A in PBS]. The cells were filtered to remove clumps and the

stained cells were analyzed using a FACS Aria II flow cytometer

(FACSDiva v5 software, Becton Dickinson). Cells transfected with the

empty pcDNA vector served as controls.

Real-time analysis of the EMT markersTotal RNA from transfected cells were extracted using Trizol (Sigma).

The RNA samples were quantified using the ND-1000 spectrophotometer

(NanoDrop Technologies, Wilmington, DE). cDNA from 100 ng of total

RNA was prepared using SuperscriptH VILO cDNA Synthesis Kit, and

quantitative PCR amplification was performed with Power SYBRHGreen

PCR Master Mix (Life Technologies, CA). Oligonucleotides were

designed using Primer Express version 3 and were purchased from

Ocimum Biosolutions. The relative expression was calculated using the

DDCt method using 18S rRNA as the endogenous control. Estrogen-

treated untransfected cells served as the calibrator. The run was

performed on the 7900HT Fast Real-Time PCR system (Applied

Biosystems, CA) under standard cycling conditions. Melting curve

analysis was performed to ensure product specificity.

Invasion assayInvasion assays using cells that had been transfected with a vector

encoding STAT3–EGFP or MCL-1–DsRed alone, co-transfected with

STAT3–EGFP or MCL-1–DsRed, or the empty EGFP and DsRed vectors

(control) were performed using the BD BioCoat Tumor Invasion Assay

System containing the BD Falcon FluoroBlok 24-multiwell insert

according to the manufacturer’s instructions. The fluorescence was

measured using the Infinite 200 Tecan microplate reader.

Statistical analysisIn all the statistical analyses, P values were obtained by applying a two-

tailed, type 2 Student’s t-test using Microsoft Excel. P,0.05 was

considered significant.

AcknowledgementsWe gratefully acknowledge the confocal imaging core at Rajiv Gandhi Centre forBiotechnology, the technical assistance of P. Manoj in the sequencing core and V.Jiji and K. G. Anurup in confocal imaging.

Competing interestsThe authors declare no competing interests.

Author contributionsM.L. was responsible for project conception, design and supervision.A.P.R. performed experiments shown in Figs. 1–7. S.T. contributed to Fig. 1 andS.T. and R.K.J. contributed to Fig. 3 along with A.P.R., M.M. contributed to theconstruction of deletion constructs in Fig. 5B and P.N. and A.P.R. contributed tocell line work in Figs. 3–5 and Fig. 7. A.P.R. and M.L. contributed Fig. 8. M.M. wasinvolved in deletion construct creation with A.P.R. for Fig. 5. M.L., A.P.R. and S.T.wrote the paper. All authors discussed the results and commented on themanuscript.

FundingThis work was supported from grants from the Department of Science andTechnology (DST), India to Malini Laloraya [vide sanction numbers SP/SO/C-51/2000 and SR/SO/AS-30/2006] and Rajiv Gandhi Centre for Biotechnology corefunds. A.P.R. received a Junior Research Fellowship from the University GrantsCommission, New Delhi, India [sanction number 10-2(5)/2006(i)-E.U.II]. A JuniorResearch Fellowship from DSTand a Senior Research Fellowship from the IndianCouncil of Medical Research, New Delhi, India, supported S.T. [grant numbersSP/SO/C-51/2000 and 2004-04790, respectively] and fellowships from the DSTsupported M.M. and P.N. [grant number SR/SO/AS-30/2006].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.138214/-/DC1

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