Epidermal growth factor (egf)

For a mammosphere forming assay by radiation-induced ROS, cells were seeded in 10 cm plates and were incubated for 24 h for attachment. Cells were incubated with serum-reduced (0.2% FBS) medium for 16 h for starvation and irradiated with 10 Gy with or without 30-min pretreatment with 100 nM vactosertib. After 24 h of incubation, we harvested the adherent cells with trypsin–EDTA and reseeded the cells in ultra-low attachment dishes. Cells were incubated in DMEM/F-12 (1:1) (GenDEPOT) supplemented with B-27 Supplement (50X) (Gibco Laboratories), 10 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen), and 10 ng/ml Epidermal Growth Factor (EGF) (Sigma Aldrich). To measure mammosphere-forming efficiency (MSFE), the number of spheres (> 50 μm) was counted after one week. MSFE indicates the number of spheres divided by the original number of cells seeded and is presented as %.
For a mammosphere forming assay by GOX-generated ROS, cells were seeded in ultra-low attachment dishes and incubated in DMEM/F-12 (1:1) (GenDEPOT) supplemented with B-27 Supplement (50X) (Gibco Laboratories), 10 ng/ml bFGF (Invitrogen), and 10 ng/ml EGF (Sigma Aldrich). Cells were treated with 2.5 mU/ml GOX with or without co-treatment with 100 nM vactosertib. To measure MSFE, the number of spheres (> 100 μm) was counted after one week.

Cells were grown in 5% FBS and treated with either (a) Transforming growth factor beta-1 (TGFβ1) (Sigma) (10 ng/ml); (b) Epidermal growth factor (EGF) (Sigma) (50 ng/ml) plus TGFβ1 (10 ng/ml), or (c) EGF (Sigma) (20 ng/ml) plus basic fibroblast growth factor bFGF (Sigma) (10 ng/ml). Cells were grown continuously and passaged in the presence of these cytokines for at least 2 weeks before addition of SS. For long term SS treatment, the cells were passaged continuously with 1 mM SS for at least 2 weeks before harvesting for EMT marker mRNA analysis.

Experiments were performed on the MCF-10A cell line purchased from ATCC (Manassas, VA, USA). Cells were cultured in 2D in DMEM/F12 (Life Technologies, Carlsbad, CA, USA) containing 5% horse serum (New Zealand origin, Life Technologies, Carlsbad, CA, USA), 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin (all Sigma-Aldrich). Acini from MCF-10A were cultured as previously described [30 (link)]. In brief, single cells were seeded onto a Geltrex (ThermoFisher Scientific, Waltham, MA, USA) bed and cultivated for nine days in EGF-supplemented assay medium (DMEM/F12 Glutamax, 2% horse serum, 5 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 1 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin), changing the medium every three days. At day nine, EGF was eliminated from the assay medium for further cultivation, changing the medium every three days [30 (link)], while acini cultivation did not exceed 21 days.

Freshly sorted LCs (1000 cells) were resuspended in culture medium [DMEM/F12 lacking Phenol Red supplemented with 5 µg/ml insulin (Sigma), 10 ng/ml EGF (Sigma), 100 ng/ml cholera toxin (Sigma) and 5% FCS] and seeded onto 24-well plates in the presence of 5000 irradiated NIH-3T3 cells, as previously described (Sleeman et al., 2007 (link)). Five days later, colonies were fixed with 4% PFA, stained with Hematoxylin and Eosin, and counted.
For three-dimensional mammosphere assays, FACS-sorted luminal or basal cells (10,000 cells) were resuspended in culture medium [DMEM-F12 lacking phenol red supplemented with B27 (1×, Gibco), 20 ng/ml EGF (Sigma), 20 ng/ml bFGF (Gibco), 4 µg/ml heparin (Sigma), 10 µg/ml insulin (Sigma) containing 4% Matrigel] as previously described (Dontu et al., 2003 (link); Spike et al., 2012 (link)). After 15 days in culture, mammospheres were imaged using a stereomicroscope (Nikon SZM800). Three independent experiments were analyzed. For each traced organoid, the size and number of clones were measured using ImageJ software (NIH). For serial passaging, mammospheres were collected by centrifugation and incubated with 0.05% trypsin/EDTA (Gibco) to obtain a single cell suspension. Cells were replated in 4% Matrigel (BD Pharmingen) at a density of 5000 cells/ml, as described above. All cultures were maintained in a 5% CO₂ atmosphere at 37°C.

The protocol of iPSCs differentiation into iNSCs involving EB formation was based on the method presented by Yuan et al. [17 (link)]. The whole procedure can be divided into several distinct steps. At approximately 80% confluency iPSCs colonies were firstly transferred from 4 wells of 6-well cell culture plate (Nunc, Thermo Fisher Scientific) coated with Geltrex to a 6 cm non-adhesive culture dish/T25 non-adhesive culture bottle (Sarstedt, Germany) and grown in suspension culture in Embryoid Body Formation medium (DMEM/F-12 with GlutaMAX-I (1X), KnockOut Serum Replacement 20%, 2-Mercaptoethanol 100 μM, Non Essential Amino Acids 1%) for 4 days. During the next 4 days, 5 × 10⁻⁷ M retinoic acid (Sigma-Aldrich) was added daily to the suspension culture. Next, 5 to 10 EBs were plated on Poly-L-Ornithine (20 μg/ml)/Laminin (10 μg/ml) (both from Sigma Aldrich)–coated 6-well adherent culture dishes (Nunc) and grown in Neural Stem Cell Induction medium (DMEM/F12 1X, B-27 Supplement 2% both from Life Technologies), supplemented with 10 ng/ml hLIF (Merck Milipore), 2 μg/ml heparin sodium salt in PBS (STEMCELL Technologies, Canada), 20 ng/ml EGF (Sigma Aldrich), 10 ng/ml bFGF for the following 7 days. After reaching 85% confluency, 0.5–1 × 10⁶ cells were seeded on Geltrex-coated dishes with ReNcell medium (Merck Millipore) supplemented with 20 ng/ml EGF and 20 ng/ml bFGF.