Supplementary Information

Progesterone enhances branching morphogenesis in the mouse mammary gland by increased expression of Msx2

Satoh K, Hovey RC, Malewski T, Warri A, Goldhar AS, Ginsburg E, Saito K, Lydon JP, and Vonderhaar BK.

Materials and Methods

Animal treatment

A subgroup of mice was euthanized at different stages of development for gene expression analysis as described (Hovey et al., 2001).For mammary tissue from pre-pubescent mice, only the epithelial rich region was utilized. Mice were killed during puberty and as adults at estrus and diestrus as determined by vaginal appearance (Champlin et al., 1973) and confirmed by vaginal lavage (Rugh, 1967) to control for hormone-induced variation. Mice killed during gestation had been mated at 10 weeks of age and the presence of a seminal plug was considered day 0 of gestation. Litter size was standardized to 6-8 pups. Weaning was performed on day 10 of lactation and was referred to as day 0 of involution. Lymph nodes were removed from all mammary tissue at harvest. Mammary glands were snap frozen in liquid nitrogen and stored at –80°C until RNA was extracted.

Additional females were hormonally manipulated to study Msx2 expression. Mice were treated with slow release pellets (10mg) containing either cholesterol only (C; control), 5g E with cholesterol (2001:1, C:E), 5mg P with cholesterol (2000:1000, C:P) or E (5g) +P (5mg) with cholesterol (2002:1001:1, C:P:E) implanted subcutaneously as previously described (Plaut et al., 1993) for the indicated times. After an initial rapid rise in E and P concentration in the serum, the levels of these hormones decrease and remain constant at 2 – 4 times the basal level (Atwood et al., 2000). Where indicated, bilateral ovariectomy was performed on nulliparous BALB/c mice at 9 weeks of age under avertin (2, 2, 2-tribromoethanol, Aldrich Chemical Company, Milwaukee, WI) anesthesia. One week later when circulating ovarian steroid levels had declined but the mammary gland remained sensitive to hormonal stimulation (Haslam, 1988) hormonal pellets were implanted as above.

MMTV-Msx2transgenic mice:The plasmid, MMTV-Msx2, was made by cloning the full-length coding region of Msx2 (a gift from Dr. R.E. Maxson, Jr., USC) into the EcoRI site of the multiple cloning site of pcDNA3-1 after excising the CMV promoter and substituting the MMTV-LTR (Jhappan et al., 1992). Transgenic mice were generated at the NCI Frederick Cancer Research and Development Center on a FVB/N background. Founders were identified by Southern blot analysis using an Msx2 cDNA probe. Progeny were genotyped by PCR. Two independent transgenic lines that specifically overexpressed the transgene in the mammary gland were established and characterized. Both lines (designated as lines O2 and L8) displayed similar phenotypes and data shown are from L8. For whole-mount analysis of developing mammary glands, the #4 abdominal glands were spread on glass slides, fixed with Carnoy’s fixative and stained with carmine alum as described (Plaut et al., 1993).

RT-PCR

First strand cDNA primed with 250 ng oligo dT was generated from 1 g total RNA using M-MLV reverse transcriptase (Invitrogen) in a total reaction volume of 25 l with 1X first strand buffer, 10 mM DTT, 0.5 mM dNTP, 10 units of RNase Inhibitor and 200 units MMLV-RT. PCR was performed on 1-2.5 l of RT product in a 25 l reaction mixture using the Roche (Indianapolis, IN) PCR Master Kit with gene-specific primers as described in Table 1. PCR conditions for Msx2(Bell et al., 1993) and PR and GAPDH(Hovey et al., 2001) were previously described. Products were resolved on a 2% agarose gel in 1X Tris-borate-EDTA. Ethidium bromide-stained bands were quantitated using NIH Image. Gene expression was normalized to the respective level of GAPDH mRNA. The PCR conditions were optimized to ensure that amplification was in the linear phase for each primer pair. Appropriate negative controls were included for each RT-PCR reaction.

Cell culture and transfection

The NMuMG mouse mammary epithelial cell line (Owens et al., 1974)was routinely grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 5% FBS (Invitrogen), penicillin (100U/ml) /streptomycin (100 g /ml, Invitrogen) and 1 µg/ml of insulin (Sigma) and maintained at 37 °C in 5 % CO2 in a humidified environment.

The full-length murine Msx2 cDNA was cloned into pEF4/myc-His C vector (Invitrogen). Transfection of cells with expression vectors (empty vector or vector containing mouse Msx2 cDNA) was performed using FuGENE 6 (Roche, Indianapolis, IN) and transfectants were selected with zeocin (2mg/ml; Invitrogen). After 4 weeks, individual colonies were picked using cloning cylinders and thereafter cells were maintained in normal growth media containing zeocin (500 µg/ml). Expression of Msx2 mRNA from individual clones was confirmed by RT-PCR. EpH4-WT mouse mammary epithelial cells (Fialka et al., 1996) were maintained in DMEM growth medium (DMEM/GM) comprising DMEM, 10% FBS, 2mM L-glutamine (Invitrogen), 20 mM HEPES (Sigma) and penicillin (100U/ml)/streptomycin (100 g/ml). For hormone treatment experiments, cells were maintained in minimal DMEM media (DMEM/CSS) supplemented with 5% charcoal stripped serum (CSS; Gemini Bio-Products, Woodland, CA), glutamine, HEPES, and penicillin/streptomycin as above. For inhibitor studies, the Wnt inhibitor Dickkopf1 (Dkk1; R&D Systems, Minneapolis, MN) and the Bmp inhibitor, Noggin (R&D Systems), were added at the indicated concentration after cells had attached overnight. The full-length cDNA for the mouse PR was cloned into pcDNA 3.1. For stable transfection, cells were allowed to attach overnight, and then were transfected with either pcDNA-PR or pcDNA-EV (1ug/well) using FuGENE 6. Transfected cells were selected with neomycin (G418; 800 g/ml) for 4 weeks. Clones of EpH4-EV or EpH4-PR cells were thereafter maintained in DMEM/GM with G418 (400 g/ml). Expression of PR was confirmed by RT-PCR. For hormone treatments, cells were plated in DMEM/GM without antibiotics and grown for 48 h (to approximately 70-90% confluence) before rinsing with PBS and being changed to DMEM/CSS in the absence or presence of P (10-8 M or 10-6 M). Cells were then collected in TRIzol for the extraction of total RNA.

The mouse MC4-L3 mammary adenocarcinoma cell line (provided by Dr. C. Lanari, Buenos Aires, Argentina) (Lanari et al., 2001) was routinely grown in DMEM/F12 supplemented with 5% CSS. For hormone treatments, cells were seeded at 2 x 105 cells per well in 6 well dishes in triplicate and allowed to attach overnight. The indicated hormones (dissolved in ethanol) were then added; control wells received supplemental ethanol alone (never exceeding 0.1%). Cells were collected in TRIzol at the indicated times and RNA extracted for RT-PCR analysis.

Table 1. Primer sequences

Gene / Primers / Gene Position
GAPDH / 5’ tgaaggtcggtgtgaacggtttgc
3’ ccatgtaggccatgaggtccaccac / 71-1054
Msx2 / 5’ ggagcaccgtggatacaggag
3’ gagcacaggtcatggaaggg / 697-1104
PR (A&B) / 5’cccacaggagtttgtcaaactc
3’gtcatcactttttgtgaaagaggagcggc / 2383-2778

References for Supplementary Information

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