implanted bilaterally into pubertal or adult Gli2DS
mice. Mammary ductal morphology was examined
7 days after surgery. In parallel experiments,
mammary ducts in the vicinity (1 to 2 mm) of
implants were dissected and prepared as single-cell
suspensions. Cells derived from various implants
were plated in a 24-well Petri dish for 2 hours to
deplete fibroblasts, followed by collection of supernatant enriched in epithelial cells. Total RNAs
were extracted from epithelial cells and converted
to cDNAs, which were then applied to gene-specific (Bio-Rad) preamplification and subsequent qPCR analyses.
GH, estrogen, and prolactin ELISA
analyses in serum
Peripheral blood was drawn by retro-orbital bleeding in the amount of 200 ml from Gli2WT and Gli2DS
mice. Mice were allowed to recover for 5 days until
the next collection. Blood samples were kept at
ambient temperature for 30 min, followed by centrifugation at 3000 rpm for 10 min. Serum in the
supernatant was either used immediately for
ELISA analyses or aliquoted and stored at –80°C
until next use. Serum samples were diluted and
subjected to standard ELISA assays to determine
GH or prolactin levels in accordance with the
manufacturer’s protocol. Differences between genotypes are considered significant if P < 0.05. Estrogen
levels of Gli2WT and Gli2DS mice were determined
by 17b-Estradiol ELISA kit (from Enzolifesciences)
according to the manufacturer’s instructions.
Statistic analysis was performed with GraphPad
Prism v5. Data are presented as mean ± SEM,
and group differences were examined with a
two-tailed Student’s t test. P values were calculated on the basis of three independent experiments unless otherwise specified. A value of P <
0.05 is considered statistically significant.
Primers used in real-time
Primers used for preamplification of Igf1, Egr1,
mCsf, and Axin2 in cDNAs of dissected implants
were purchased from Bio-Rad. Primers used for
qPCR analyses of Col1a1, Col1a2, Col2a1, Pdgfra,
Pdgfb, and Esr1 were previously reported (50–52).
Others were purchased as Quanti Tect Primer
Assays from Qiagen. Quanti Tect primer assays used
for real-time RT-PCR include mGli2 (QT00291711),
Ghr (QT00109900), mBmp7 (QT00096026), mIgf1
(QT00154469), m Wnt2 (QT00118503), m Wnt2b
(QT00115451), mFgf7 (QT00172004), mHgf
(QT00158046), mEgr1 (QT00265846), mCsf1
(QT01164324), mIl-3Ra (QT00251293), mAxin2
(QT00126539), and mLef1 (QT00148834).
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