These results are consistent with our data. that D492, an epithelial cell line with stem cell properties, generates TDLU-like structures in 3D culture [33], [34]. D492 is thus a good model to dissect molecular mechanisms regulating branching morphogenesis. We have also shown that endothelial cells stimulate growth and morphogenesis of breast and lung epithelial cells [35], [36]. Most recently, we demonstrated that endothelial cells facilitate Harringtonin branching morphogenesis of D492 in co-culture and furthermore induces a subpopulation of D492 to generate spindle-like colonies through an EMT conversion [37]. Here, we show that SPRY2 is predominantly expressed in luminal epithelial cells of duct and lobuli in human breast tissue. We also show that SPRY2 is highly expressed in the pregnant and lactating mouse mammary gland with phosphorylated SPRY2 peaking during pregnancy. Expression of SPRY2 is associated with expression of phosphorylated EGFR (pY1068) and activation of the downstream MAPK signaling pathway. Using D492, we show that SPRY2 is expressed at the branching tips and suppression of SPRY2 through shRNA gene knockdown increases branching morphogenesis and promotes epithelial to mesenchymal transition when cultured with endothelial cells. Materials and Methods Cell culture The breast epithelial stem cell line D492 was maintained in H14 medium [38], consisting of DMEM/F12, 50 IU/ml penicillin, 50 g/ml streptomycin (Invitrogen), 250 ng/ml insulin, 10 g/ml transferrin, 2.6 ng/ml sodium selenite, 0.1 nM estradiol, 0.5 Harringtonin g/ml hydrocortisone, 5 g/ml prolactin (SIGMA) and 10 ng/ml EGF (Peprotech). Primary LEPs and MEPs were maintained on CDM3 and CDM4 as previously described [35], [39]. Primary human BRENCs were isolated from breast reduction mammoplasties Harringtonin as previously described [40] and cultured on endothelial growth medium (EGM-2) (Lonza) +5% FBS (Invitrogen). Preparation of 3D mono- and co-cultures 3D monocultures were carried out in 96 well culture plates (Becton Dickinson, BD, Falcon). 7103, 1104 and 1.3104 D492 cells were suspended in 300 l of reconstituted basement membrane (rBM) purchased as matrigel (BD). Co-culture experiments were carried out with 1103 D492 mixed with 5104 BRENCs. 100 l of mixed cells / rBM were seeded in each well in a 96 well plate and cultured on H14 (Monoculture) or EGM5 (Co-culture) for 16 days. Isolation and processing of mammary glands and 3D cell cultures Human tissue from breast reductions was used Harringtonin for immunohistochemistry and for isolation of primary breast epithelial cells. Primary LEPs and MEPs were isolated by magnetic cell sorting (MACS) as previously described [39]. Murine mammary glands were dissected Harringtonin from C57BL/6 mice at the following stages: 6 week old virgins, day 15 of pregnancy and day 2 of lactation. Mammary glands were snap frozen in liquid nitrogen and preserved at C80C. Isolation of colonies from 3D cell culture was done as previously described by gentle dissociation in PBS-EDTA buffer [41]. Immunochemistry Formalin-fixed, paraffin embedded human tissue blocks from reduction mammoplasty biopsies were cut into 5 m serial sections and mounted on slides. Sections were deparaffinized and rehydrated in xylene and ethanol. Antigen retrieval was done by boiling in EDTA buffer for 15 minutes. Frozen mouse mammary glands were cryosectioned at 15 m setting following formalin fixation. The following primary antibodies were used; Sprouty-2 (#07-524, Upstate/Millipore), CD-31 Mouse monoclonal antibody to Pyruvate Dehydrogenase. The pyruvate dehydrogenase (PDH) complex is a nuclear-encoded mitochondrial multienzymecomplex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), andprovides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle. The PDHcomplex is composed of multiple copies of three enzymatic components: pyruvatedehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase(E3). The E1 enzyme is a heterotetramer of two alpha and two beta subunits. This gene encodesthe E1 alpha 1 subunit containing the E1 active site, and plays a key role in the function of thePDH complex. Mutations in this gene are associated with pyruvate dehydrogenase E1-alphadeficiency and X-linked Leigh syndrome. Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene (M0823, DakoCytomation), Keratin 19 (ab7754, Abcam), Keratin 14 (NCL-LL002, NovoCastra), PCNA (ab29, Abcam), EGFR (#4267, Cell Signaling), p-EGFR (Tyr1068) (#3777, Cell Signaling), ki67 (Abcam, ab833), E-Cadherin (BD Biosciences, cat. 610182), N-Cadherin (BD Biosciences, cat. 610921).Fluorescent nuclear counterstain, TO-PRO-3 (Invitrogen) was used in immunofluorescence. Specimens were visualized on a Zeiss LSM 5 Pascal laser-scanning microscope (Carl Zeiss). In situ Proximity Ligation assay Protein phosphorylation of Spry2 was studied by Proximity Ligation assay (PLA) using the Duolink(R) kit (Olink Bioscience, Uppsala, Sweden) [42]. Sections from mouse mammary glands and 3D.

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