The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Open access This work is licensed under the Creative Commons Attribution 4.0 License. Cyclosporin A or in a scaffold-free manner . By adopting this acinus-like structure composed of multiple cell types, it uses complex matrices and scaffolds, whereby myoepithelial cells are absent. Bilayered acini composed of a mix of purified human luminal and myoepithelial cells isolated from normal mammary glands have also been developed, with or without fibroblasts [10,39]. However, because they are formed using primary cultures, these models have some limitations. For instance, they are hardly genetically manipulable, access to human tissues is difficult, and there is a great patient-to-patient variability. There is thus still a great need for a Cyclosporin A simplified, optimized, genetically manipulable, reproducible and physiologically relevant model to recapitulate the normal structure of the functional unit of the human mammary gland C the bilayered acinus . By using commercially available cell lines instead of primary cells, such a model will be accessible to the scientific community and allow more mechanistic studies in the understanding of the biology of the human Gdf11 bilayered acini. This study aimed to develop a model representing the breast bilayered acini that can be genetically manipulated and easily reproduced by using cell lines. Here, two combinations of nontumorigenic cell lines were investigated: the human luminal and myoepithelial-like cells MCF-12A and Hs 578Bst; and the murine luminal and myoepithelial cells SCp2 and SCg6 . Materials & methods Cell lines MCF-12A cells (ATCC? CRL-10782) and Hs 578Bst cells (ATCC HTB-125) were purchased at ATCC (ATCC, VA, USA). SCp2 and SCg6 cells were a gift from Calvin Roskelley (University of British Columbia [UBC]). MCF-12A cells were maintained in phenol red-free Dulbecco’s modified Eagle’s medium Ham’s F12 (DMEM/F12) culture medium (21041025, ThermoFisher Scientific, IL, USA) supplemented with 5% (v/v) horse serum (ThermoFisher Scientific, 16050C122), hEGF recombinant (20?ng/ml) (PHG0311, Invitrogen, Cyclosporin A MA, USA), hydrocortisone (500?ng/ml; H0888, Sigma-Aldrich, Oakville, Ontario, Canada), insulin (10?g/ml; Sigma-Aldrich, C8052), cholera toxin (100?ng/ml; Invitrogen, 12585014) and propagated according to ATCC guidelines. Hs 578Bst cells were maintained in Hybri-Care medium (ATCC 46-X?) supplemented with 10% (v/v) activated fetal bovine serum (098150, Wisent Bioproducts, Saint-Jean-Baptiste, Quebec, Cyclosporin A Canada) and mouse EGF (EGF from murine submaxillary gland, 30?ng/ml; Sigma-Aldrich, E4127) and propagated according to ATCC guidelines. SCp2 and SCg6 cells were grown in DMEM/F12 medium (Sigma-Aldrich, D2906) supplemented with insulin (5?g/ml) and fetal bovine serum (5% v/v). Western blot analysis Cell monolayers were washed twice with phosphate buffered saline (PBS) before the addition of lysis buffer (Tris 50?mM, NaCl 150?mM, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P40, 0.5% sodium deoxycholate, pH 8) supplemented with NaF 1.25?M, NaVO3 1?M and Halt Protease and phosphatase cocktail inhibitor (Fisher Scientific, Ontario, Canada). Cells were scraped, collected and incubated on ice for 5?min. Cell lysates were centrifuged for 10?min at 2500?r.p.m. at 4C. The supernatants were aliquoted and stored at -80C until further Cyclosporin A processing. Lysate protein concentrations were measured using a bicinchoninic acid protein assay reagent kit (Thermo Scientific #23227). Protein samples were resolved on stain-free acrylamide gels (TGX Stain-Free FastCast Acrylamide kit, 10%, Bio-Rad, Ontario, Canada) and transferred onto polyvinylidene fluoride membranes?(PVDF). Membranes were clogged with Tris Buffered Saline (TBS)-Tween 20 (0.1%; Fisher Scientific) comprising 3% bovine serum albumin (BSA) or dry milk, relating to manufacturer instructions, for 1?h and incubated overnight at 4C with the following main antibodies: anti-E-cadherin (#3195; Cell Signaling Technology, MA, USA), anti-Cytokeratin 18 (K18) (#ab52948; Abcam, MA, USA), anti-Cytokeratin 14 (K14) (#MS-115-P1ABX; Thermo Scientific, Cheshire, UK), anti-alpha-Smooth muscle mass actin (-SMA) (#M0851; Dako, Glostrup, Denmark), anti-calponin-1 (#17819; Cell Signaling Technology), anti-caldesmon-1 (#12503; Cell Signaling Technology) and anti-p63 antibody (#13109; Cell Signaling Technology). Bound main antibody was recognized using HRP-conjugated.