Development of mammary
luminal progenitor cells is
controlled by the transcription
Daisuke Yamaji,1Risu Na,1Yonatan Feuermann,1
Susanne Pechhold,2Weiping Chen,3Gertraud
W. Robinson,1,5and Lothar Hennighausen1,4
1Laboratory of Genetics and Physiology, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, Maryland 20892, USA;2Diabetes Branch,
National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland
20892, USA;3Genomics Core Laboratory, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, Maryland 20892, USA
Mammary alveologenesis is abrogated in the absence of
the transcription factors STAT5A/5B, which mediate
cytokine signaling. To reveal the underlying causes for
this developmental block, we studied mammary stem
and progenitor cells. While loss of STAT5A/5B did not
affect the stem cell population and its ability to form
mammary ducts, luminal progenitors were greatly re-
duced and unable to form alveoli during pregnancy.
Temporally controlled expression of transgenic STAT5A
in mammary epithelium lacking STAT5A/5B restored the
luminal progenitor population and rescued alveologenesis
in a reversible fashion in vivo. Thus, STAT5A is neces-
sary and sufficient for the establishment of luminal
Supplemental material is available at http://www.genesdev.org.
Received July 6, 2009; revised version accepted September 1,
STAT5A/5B, are two highly conserved transcription fac-
tors activated by various cytokines—including prolactin,
growth hormone, and EGF—that play important roles in
the development and function of mammary glands,
hematopoietic cells, liver, and muscle (Hennighausen
and Robinson 2008). Deletion of the Stat5a gene in the
germline results in impaired mammary alveologenesis
duringpregnancy (Liuet al.1997). However, inthe absence
of Stat5a, increased expression of STAT5B after several
pregnancies partially rescued the lactation defect (Liu
et al. 1998). Deletion of Stat5b had no effect on mammary
epithelial cells (Udy et al. 1997; Teglund et al. 1998). In
subsequent studies using germline deletion, as well as
and STAT5B, collectivelyreferredto as
mammary epithelial-specific ablation, of both Stat5a and
Stat5b genes, the role of STAT5A/5B during mammary
gland development was investigated (Miyoshi et al. 2001;
Cui et al. 2004). We discovered that the deletion of both
Stat5a/5b genes in mammary epithelium resulted in
a severe defect of alveologenesis, and that the presence
of STAT5A/5B was essential for the proliferation, differ-
entiation, and survival of mammary epithelial cells.
Mammary epithelium consists of two types of cells:
basal myoepithelial cells and luminal cells, which form
a ductal tree in virgins and alveoli during pregnancy.
These events are coordinated by systemic hormones and
cytokines (Hennighausen and Robinson 2005). Elabora-
tion of mature epithelium from stem cells is thought to
proceed in a hierarchical progression. Stem cells give rise
to transient amplified progenitor cells capable of gener-
ating ductal and alveolar structures that become re-
stricted to only ductal or alveolar fates and eventually
give rise to differentiated lineages (Stingl 2009). In recent
years, a combination of enzyme digestion and fluores-
cence-activated cell sorting (FACS) techniques have been
developed to allow the isolation of these different cell
populations from single-cell suspensions derived from
mammary tissue of virgin female mice (Shackleton
et al. 2006; Stingl et al. 2006). This knowledge enabled
us to study the role of STAT5A/5B in a defined cell
population of mammary epithelium.
STAT5A/5B control stem and progenitor cell fate in the
hematopoietic system (Wang et al. 2009). In the absence
of STAT5A/B, mice fail to develop T, B, and natural killer
cells (Hoelbl et al. 2006; Yao et al. 2006). STAT5A/5B are
also required for the maintenance and expansion of
primitive stem and progenitor cells, both in normal and
leukemic hematopoiesis (Li et al. 2007; Liu et al. 2008).
These studies support our proposal that STAT5A/5B are
critical for mammary cell lineage development from
primitive stem/progenitor cells.
Several mechanisms might account for the lack of
alveolar development in the absence of STAT5A/5B: (1)
Stem cells are defective and fail to generate alveolar
progenitor cells. (2) Although stem cells generate alveolar
progenitor cells, progenitors cannot proliferate or survive.
(3) Although stem cells give rise to alveolar progenitor
cells that can proliferate and survive, progenitors do not
generate daughter alveolar cells. (4) STAT5A/B play a role
only in differentiated alveolar cells. To test these hypoth-
eses, we isolated and analyzed epithelial stem and pro-
genitor cell populations from mammary epithelium con-
taining or lacking STAT5A/5B.
Results and Discussion
The mammary luminal progenitor cell population
is reduced in the absence of STAT5A/5B
To ask which of the steps in the lineage progression of
mammary stem cells to functional secretory epithelium
is dependent on the presence of STAT5A/5B, we used
conditional gene deletion with a MMTV-Cre transgenic
mouse line that affects all epithelial cells of the newborn
as determined with a lacZ reporter construct. Therefore,
we consider the entire epithelium null for Stat5a/5b
(Wagner et al. 1997, 2001; Buono et al. 2006). Our
observation that Stat5a/5bfl/fl;Cremice could not lactate
[Keywords: STAT5; alveologenesis; mammary gland; progenitor; CD61;
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1840109.
2382GENES & DEVELOPMENT 23:2382–2387 ISSN 0890-9369/09; www.genesdev.org
even after five to six pregnancies further supports this.
We prepared single-cell suspensions from mammary
tissue of nulliparous mature female Stat5a/5bfl/fland
et al. 2006; Asselin-Labat et al. 2007). Both Stat5a/5bfl/fl
and Stat5a/5bfl/fl;Crevirgin mice showed similar dot plot
patterns that defined four cell populations: negative
(CD24?CD49f?), luminal (CD24hiCD49flo), myoepithelial
(CD24loCD49fhi), and the stem cell-enriched upper portion
of myoepithelial cells (CD24midCD49fhi) (Fig. 1A). Percent-
ages of each population in Stat5a/5bfl/flmice versus Stat5a/
5bfl/fl;Cremice were 11.1% 6 0.97% versus 11.5% 6 1.33%
in luminal, 19.0% 6 3.06% versus 11.3% 6 1.82% in
myoepithelial, and 3.38% 6 0.33% versus 2.00% 6 0.40%
in stem cell-enriched populations, respectively. Quantita-
tive real-time RT–PCR analysis revealed that Stat5a and
Stat5b mRNAs were not present in the luminal, myoepi-
thelial, and stem cell-enriched fractions in mammary
tissue of Stat5a/5bfl/fl;Cremice (Supplemental Fig. 1).
Stat5a/5b mRNAs were present in the CD24?CD49f?
population in both Stat5a/5bfl/fland Stat5a/5bfl/fl;Cremice,
suggesting that these were of stromal origin. These results
corroborate the high deletion efficiency in this MMTV-Cre
transgenic line (Wagner et al. 1997, 2001; Buono et al.
To assess the ability of mammary stem cells to form an
entire functional mammary gland in the presence or
absence of STAT5A/5B, the FACS-purified stem cell-
enriched populations were transplanted into cleared fat
pads of athymic nude mice. Mammary stem cells from
Stat5a/5bfl/fland Stat5a/5bfl/fl;Cremice were able to
generate ductal outgrowths with primary and secondary
side branches (Supplemental Fig. 2A,B). This is in agree-
ment with the results obtained by transplantation of
small pieces of mammary tissue from Stat5a/5bDN/DN,
another mouse line with a partially disrupted Stat5a/5b
locus (Miyoshi et al. 2001). Limiting dilution analyses
revealed no difference in the ability of stem cells to
generate epithelial outgrowths in the presence or absence
of STAT5A/5B (Supplemental Table 1). These results
demonstrate that STAT5A/5B are not important for the
ability of mammary stem cells to reconstitute both
luminal and myoepithelial cell lineages in ducts of virgin
mice. To assess alveolar development and differentiation,
stem cell-transplanted hosts were mated to induce preg-
nancy. While Stat5a/5bfl/flstem cells were able to de-
velop a full alveolar compartment at term (Supplemental
Fig. 2C,E), the epithelial compartment in Stat5a/5bfl/fl;Cre
stem cell transplants remained sparse (Supplemental
Fig. 2D,F). Histological analyses demonstrated that out-
sisted of cells secreting milk (Supplemental Fig. 2G,I), while
the Stat5a/5bfl/fl;Crestem cells produced only a ductal tree
with virgin-like characteristics (Supplemental Fig. 2H,J).
These results validate earlier studies that alveolar de-
velopment is inhibited in the absence of STAT5A/5B.
To determine potential causes for the absence of
alveolar development, we analyzed luminal progenitor
cells. The amount of CD61+cells in the luminal cell
population was profoundly reduced in Stat5a/5bfl/fl;Cre
mice (44.4% 6 3.93% in Stat5a/5bfl/flvs. 25.8% 6 0.81%
in Stat5a/5bfl/fl;Cre) in virgins (Fig. 1B). Furthermore, the
CD61+population was not increased during pregnancy
(Fig. 1B,C). This strongly suggests thatthe CD61+luminal
progenitor cells present in the Stat5a/5bfl/fl;Cremammary
gland are ductal luminal progenitor cells, and the re-
duction in the total number of CD61+cells reflects an
absence of alveolar precursors. Thus, impaired alveolo-
genesis and lactation of Stat5a/5b-null mammary epithe-
lium most likely are due to a reduced number of alveolar
luminal progenitor cells already in the virgin state.
STAT5A is necessary and sufficient for the generation
of alveolar luminal progenitor cells and mature alveoli
We next asked whether the deficit in CD61+cells in the
ducts of virgin glands and the failure to generate alveoli
can be overcome by a temporally defined expression of
STAT5A after the ductal system has been established at
transgene under control of the tetracycline-inducible
5bfl/flmice (left panels) and Stat5a/5bfl/fl;Cremice (right panels). (A)
Dot plot pattern of luminal (Lum; CD24hiCD49flo), myoepithelial
(Myo; CD24loCD49fhi) and stem cell-enriched (Stem; CD24midCD49fhi)
populations in 12-wk-old virgin mice. (B) CD61 staining histograms
of luminal cell population (CD24hiCD49flo) in the mammary gland
of 12-wk-old virgin mice (top panels) and 6 d pregnant mice (P6,
bottom panels). The CD61+population was determined according to
fluorescence minus one (FMO) control of the same mouse. (C) Bar
graph depicting the percentage of CD61+cells in luminal cell pop-
ulations of Stat5a/5bfl/fl(blank bars) and Stat5a/5bfl/fl;Cre(filled bars)
mammary glands in virgins, and on day 6 (P6) and day 12 (P12) of
pregnancy. Values are means 6 SD. (*) P < 0.05.
Flow cytometry analyses of mammary cells from Stat5a/
STAT5 and mammary alveologenesis
GENES & DEVELOPMENT2383
promoter (tet-op-Stat5a), as well as MMTV-rtTA (Gunther
et al. 2002) and MMTV-Cre transgenes and two floxed
Stat5a/5b alleles (Stat5a/5bfl/fl;Cre;iStat5). These experi-
mental mice were fed with doxycyline-containing food
to induce transgenic expression of Stat5a and were
continuously mated. Mice being exposed to doxycycline
during pregnancy developed functional mammary glands
during pregnancy and were able to feed their pups. In
order to demonstrate the dependence of mammary de-
velopment and lactation on the expression of transgenic
STAT5A induced by doxycycline, successfully nursing
dams were switched to regular food after weaning of the
second litter. Insufficient amounts of milk were present
and pups of the third and fourth litters did not survive.
Return to doxycycline food, and therefore expression of
transgenic Stat5a, after the death of the fourth litter
resulted in the survival of two more litters.
Mammary tissue samples from the Stat5a/5bfl/fl;Cre;iStat5
mice taken by biopsy in the morning after delivering the
first, fourth, and sixth litters were analyzed by histology
and immunofluorescence staining (Fig. 2A). Expanded
alveoli lined with differentiated secretory cells were found
after doxycycline treatment (Fig. 2B, panel a). The lumen
contained proteinaceous material and lipid droplets. In
the absence of doxycycline, the alveoli were small, secre-
tory cells appeared undifferentiated, and the lumina were
unexpanded and devoid of lipid droplets (Fig. 2B, panel b).
Activated STAT5 was found inthe majority of nuclei after
doxycycline treatment, but was scarce in the absence of
doxycycline (Fig. 2B, panels d,e). Epithelial differentiation
was reversed when expression of exogenous STAT5Awas
abolished and could be induced again upon stimulation of
STAT5A expression (Fig. 2B, panels c,f). Furthermore,
staining of the sodium/potassium/cotransporter 1 (NKCC1),
a protein expressed in undifferentiated ductal cells of
virgin and early pregnancy stages (Shillingford et al.
2002), was seen in few luminal cells in the samples from
the first and sixth lactation, but was strongly expressed in
the absence of doxycycline after the fourth pregnancy
(data not shown). These results show that the presence of
only STAT5A during pregnancy leads to the formation of
alveolar structures and functional differentiation of lu-
minal cells. It clearly indicates that Stat5a/5b-deficient
mammary epithelium contains stem cells that are capa-
ble of differentiation along the ductal but not alveolar
luminal cell lineage, and luminal cell fate depends on
To evaluate the role of STAT5A in the generation of
alveolar luminal progenitor cells, flow cytometry analy-
ses were performed with mammary tissues of Stat5a/
5bfl/fl;Cre;iStat5mice 4 wk after weaning (Fig. 3). When fed
able to generate CD61+luminal progenitor cells during
pregnancy to a similar extent as Stat5a/bfl/flmice. In con-
trast, the number of CD61+luminal progenitor cells in the
mammary glands of Stat5a/bfl/fl;Cre;iStat5mice fed with
normal diet was comparable with that of Stat5a/5bfl/fl;Cre
mice. These results demonstrate that expression of
STAT5A in the Stat5a/5b-deficient mammary epithelium
was necessary and sufficient for the generation of luminal
progenitor cells in the quiescent gland and alveologenesis
We performed genome-wide microarray analyses to
investigate changes in the gene expression profile of
mary tissues on day 6 of pregnancy. We
considered this a stage that would allow
the identification of genes that are in-
volved in the early stages of luminal cell
proliferation and differentiation and are
induced by STAT5. Besides b-casein, cyclin
D1, and connexin 26 (Hennighausen and
Robinson 2005), which are known STAT5
target genes, Gene Ontology analyses
showed decreased expression of genes
involved in regulation of the cell cycle,
DNA metabolic processes, response to
DNA damage stimuli, and chromosome
organization pathways in Stat5a/5b-
deleted samples (Supplemental Table 2).
These genes may be either directly tran-
scribed by STAT5 or represent targets of
genomic regulatory networks activated
by STAT5 in the early phase of pregnancy
that is characterized by proliferation and
rapid expansion of cells.
Impaired mammary development also
occurs in Gata3- and Elf5-deficient mam-
mary epithelium (Asselin-Labat et al.
2007; Oakes et al. 2008). To explore a
possible link between these transcription
factors and STAT5A, we measured the
expression of these two genes in the stem
and progenitor cell fractions. mRNA
levels of Gata3 were similar in both cell
populations, irrespective of the genotype
(Supplemental Fig. 3). However, expression
absence of doxycycline. (A) Schematic design of the experimental protocol. Mice were fed
with doxycycline-containing food from mating to the end of the second lactation, and again
after delivery of the fourth litter. (B) Mammary glands of Stat5a/5bfl/fl;Cre;iStat5mice were
biopsied on the day of delivering the first (panels a,d), fourth (panels b,e), and sixth (panels
c,f) litters and were stained with hematoxylin and eosin (panels a–c) or immunostained
with anti-phosphorylated STAT5 antibody (red) and anti-E-cadherin antibodies (green)
(panels d–f). Black arrows indicate lumina. White arrows point to cells containing nuclear
Histology of mammary tissue of Stat5a/5bfl/fl;Cre;iStat5mice in the presence or
Yamaji et al.
2384GENES & DEVELOPMENT
of Elf5 was barely detectable in the Stat5a/5bfl/fl;Crecells
(Supplemental Fig. 3).
This demonstrates a distinct function for these tran-
scription factors in the different cell lineages (Fig. 4).
GATA3, which hasbeen shownto controlmaintenanceof
luminal cells in ducts as well as alveoli (Kouros-Mehr
et al. 2006; Asselin-Labat et al. 2007), is not regulated by
STAT5A/5B. A higher proportion of CD61+luminal pro-
genitor cells in Gata3-deficient mammary tissues is
observed in virgins and sustained through pregnancy,
reflecting a block of differentiation to mature ductal and
alveolar luminal cells (Asselin-Labat et al. 2007). Elf5-null
mammary epithelium also contains a larger fraction of
CD61+luminal progenitor cells during pregnancy but not
in virgins, indicating a block of differentiation to secre-
tory alveolar cells (Oakes et al. 2008). We observed a de-
crease of CD61+luminal progenitor cells in the Stat5a/
5b-null mammary epithelium already in virgins and no
increase of this cell population during pregnancy. Since
ductal morphogenesis is not affected, this indicates that
STAT5A/5B regulate the production or expansion of
alveolar luminal progenitor cells (Fig. 4). The current lack
of surface markers to distinguish ductal and luminal
progenitor cells makes it impossible to discriminate the
two possibilities. Our results indicate a failure of primi-
tive stem/progenitor cells to generate alveolar luminal
progenitor cells in the absence of STAT5A/5B.
We previously identified Elf5 as a gene highly up-
regulated in lactating mammary tissue (Renou et al. 2003).
Like STAT5A/5B, ELF5 is a mediator of the prolactin–
receptor signaling pathway (Harris et al. 2006). ELF5
binding to the proximal Stat5a gene promoter in late
pregnancy has been demonstrated, and loss of ELF5 in
primary mammary epithelial cells resulted in a modest
decrease in the overall levels of STAT5A (Choi et al.
2009). Real-time RT–PCR analyses demonstrated that the
levels of Elf5 mRNA were lower in Stat5a/5bfl/fl;Cre
compared with Stat5a/5bfl/fl, and that Elf5 mRNAwas in-
duced to a greater extent by prolactin in mammary tissue
of Stat5a/5bfl/flmice as compared with Stat5a/5bfl/fl;Cre
mice (Supplemental Fig. 4), indicating cell- and stage-
specific cross-regulation of their expression in the course
of mammary development. In addition, we found 48 sites
of TTCNNNGAA, the consensus STAT5-binding se-
quence, in the distal region of the Elf5 gene promoter.
The closest one to the transcription start site is TTCA
GTGAA at 3727 base pairs (bp) upstream. Among the 48
sites, three (at ?111,201 and ?96,996, at ?65,696 and
?62,683, and at ?51,935 and ?51,189 in mice and hu-
mans, respectively) plus their flanking sequences are well
conserved between humans and mice. This indicates that
prolactin-induced Elf5 expression is regulated bySTAT5A/
5B-dependent as well as STAT5-independent mechanisms.
Together, induction of Elf5 by prolactin is partially regu-
lated by STAT5A/5B for the proliferation and differentia-
tion of mammary alveolar cells during pregnancy. How-
ever, these data are obtained from whole mammary tissue
samples rather than enriched cell populations. We found
that Stat5a/5b-null luminal progenitor cells did not ex-
press Elf5 (Supplemental Fig. 3). The presence of alveolar
luminal progenitor cells in Elf5-null mammary epithelium
in virgins puts STAT5A/5B above ELF5 in the gene hi-
erarchy (Oakes et al. 2008). It further suggests that the
absence of Elf5 expression in the Stat5a/5b-null CD61+
progenitor cells reflects the paucity of alveolar progenitor
cells in the Stat5a/5b-null mammary epithelium.
other transcription factors in the development of mammary epithe-
lial cell lineages. (Top row) In the wild-type mammary gland,
epithelial stem cells generate alveolar luminal progenitor cells that
develop mature alveoli during pregnancy. (Middle) Alveolar pro-
genitor cells fail to differentiate into secretory luminal cells in the
absence of GATA3 or ELF5, leading to their accumulation in
pregnancy and lack of alveolar development. (Bottom row) Loss of
STAT5A/5B impairs development of alveolar precursor cells (this
study) as well as maintenance of mature alveolar cells as shown
Schematic model depicting the role of STAT5A/5B and
cells in the presence or absence of STAT5A/5B. Dot plot patterns of
CD24 and CD49f populations and histograms of CD61 staining of
luminal cell population in the mammary tissue of mice with indicated
genotype 4 wk after weaning. Mammary tissues of Stat5a/5bfl/fl;Cre;iStat5
mice were analyzed with or without feeding doxycycline.
Flow cytometry analyses of mammary luminal progenitor
STAT5 and mammary alveologenesis
GENES & DEVELOPMENT 2385
The reversible generation of luminal progenitor cells
and the rescue of alveologenesis by the transgenic ex-
pression of STAT5A prove that STAT5A is able to induce
the development of the alveolar progenitor lineage in
Stat5a/5b-null mammary stem cells during pregnancy,
even if STAT5A/5B were not present during the ductal
development period during puberty. Indeed, mammary
alveologenesis was impaired by the germline deletion of
Stat5a gene but not Stat5b (Liu et al. 1997; Udy et al.
1997; Teglund et al. 1998). Taken together with our
previous findings that STAT5A/5B are critical for the
survival and function of alveolar cells during pregnancy
(Cui et al. 2004), this supports the notion that STAT5A is
required not only for the proliferation or survival of
alveolar cells, but also the generation of alveolar pro-
genitors from stem cells. This study provides evidence for
a mechanism by which the normal mammary epithelial
cell hierarchy is established and maintained through the
transcription factor STAT5A.
Materials and methods
Mouse and genotype analysis
All animals were handled and housed in accordance with the guidelines of
the National Institutes of Health Animal Care and Use Committee.
Generation of MMTV-Cre and MMTV-rtTA transgenic mice (kind gift
from Dr. L. Chodosh) and Stat5a/bfl/flmice was described previously
(Wagner et al. 1997; Gunther et al. 2002; Cui et al. 2004). We mated
Stat5a/bfl/flmice with the MMTV-Cre transgenic mouse line A in order
to generate Stat5a/bfl/fl;Cremice. The presence of Cre recombinase it-
self did not affect the number of luminal progenitor cells (Supplemental
Generation of iStat5 (MMTV-rtTA;tet-op-Stat5a)
Tetracycline-inducible STAT5A transgenic mice were generated by in-
troducing a SacII site into Stat5a cDNA at the 59 ATG start codon and
a XbaI site at the 39 TGA stop codon by PCR, using MSCV-IRES-Stat5a as
(forward) and 59-ATATCTAGATCAGGACAGGGAGCTT-39 (reverse). The
purified SacII and XbaI fragment was subcloned into plasmid p43 (kindly
provided by Dr. Priscilla A Furth), which is composed of the tet operator
sequences followed by a CMV minimal promoter and the rabbit b-globin
intron and poly(A) signal at the 39 end. The NotI-released DNA fragments
were injected into fertilized C57BL/6 eggs (The Jackson Laboratory) to
generate transgenic mice according to standard procedures. For PCR
analysis of the founders, a primer set 59-CTGAGTTCGTCAATGCATC
CA-39 (forward) and 59-GGTGATACAAGGGACATCTT-39 (reverse) was
designed to detect the unique area of the tet-op-Stat5a. The PCR protocol
was 35 cycles of 30 sec at 94°C, 45 sec at 57°C, and 1 min at 72°C. An
amplified product of 411 bp from tet-op-Stat5a was detected.
Preparation of single cells from mammary gland
Single cells from mammary tissue were prepared as described previously
(Stingl et al. 2006) with minor modifications. Briefly, mammary tissue
from virgin female Stat5a/bfl/flmice and Stat5a/bfl/fl;Cremice at 12–16 wk
of age were digested for 8 h at 37°C in complete EpiCult-B medium
(EpiCult-B medium with 5% fetal bovine serum, 10 ng/mL recombinant
human epidermal growth factor, 10 ng/mL recombinant human basic
fibroblast growth factor, 0.0004% Heparin) supplemented with 300 U/mL
collagenase and 100 U/mL hyaluronidase. After lysis of red blood cells in
NH4Cl, asingle-cell suspension was obtainedby sequential dissociation of
the fragments with prewarmed 0.25% trypsin-EDTA for 1–2 min, fol-
lowed by prewarmed 5 mg/mL dispase II plus 0.1 mg/mL DNase I (DNase;
Sigma) for 2 min, and filtration through a 70-mm mesh. All reagents were
from StemCell Technologies, Inc. unless otherwise specified.
FACS analysis and cell sorting
Prepared single cells were stained with biotinylated anti-CD45, anti-
Ter119, and anti-CD31 antibody (StemCell Technologies); anti-CD24-
fluorescein isothiocyanate (FITC, clone M1/69, BD Biosciences); anti-
CD49f-R-phycoerythrin (R-PE, clone GoH3, BD Biosciences); anti-CD61-
Alexafluor647 (clone 2C9.G2, BioLegend); and 7-amino-actinomycin D
(7AAD, BD Biosciences), followed by pacific blue-conjugated streptavidin
(Invitrogen). FACS analysis and cell sorting were performed using Diva
version 6.1.1 software and FACSAria (BD Biosciences).
Harvested mammary tissues were fixed in 10% formalin, dehydrated
through ethanol and xylene, embedded in paraffin, and sectioned. For
immunostaining, antigen unmasking was performed in a Decloaking
chamber (Biocare Medical) using BORG Decloaker Solution (pH 9.5)
(Biocare Medical) for 5 min at 125°C, 18–24PSI. The sections wereblocked
for 30 min in TBS-T containing 3% goat serum. Primary antibodies were
incubated overnight at 4°C (phosphorylated Stat5, Cell Signaling Tech-
nologies, #9314, 1:200; E-cadherin, BD Biosciences #610182, 1:200).
AlexaFluor488- or AlexaFluor594-conjugated secondary antibodies (Invi-
trogen) were used at a dilution of 1:400 for 30 min at room temperature.
Weare grateful to Dr.Lewis Chodosh for MMTV-rtTA transgenic mice, Dr.
Priscilla Furth for plasmid p43, and all the members of the Laboratory of
Genetics and Physiology for helpful discussions. This research was
supported by the Intramural Research Program of the National Institute
of Diabetes and Digesitve and Kidney Diseases, NIH.
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