P00033

All cell lines and the materials used for cell culture were ordered from Procell (Wuhan, China, https://www.procell.com.cn/) unless specified otherwise. Human breast epithelial cell MCF-10A (CL-0525) and BC cell lines MCF-7 (CL-0149), MDA-MB-231 (CL-0150), MDA-MB-436 (CL-0383), and SK-BR-3 (CL-0211) were ordered and cultured as needed.
MCF-10A cells were maintained in Dulbecco’s modified Eagle’s medium/F12 medium (PM150312) blended with 5% horse serum (164215), 20 ng/mL of epidermal growth factor (P00033, Solarbio Lifesciences, China), 0.5 μg/mL of hydrocortisone (G8450, Solarbio Lifesciences, China), 10 μg/mL of insulin (I8830, Solarbio Lifesciences, China), 1% non-essential amino acid (PB180424), and 1% penicillin−streptomycin (PB180120). MCF-7 cell was cultured in minimum essential medium (PM150140) with 0.01 mg/mL of insulin. Leibovitz’s L-15 medium (PM151010) was used to ensure the growth of MDA-MB-231 and MDA-MB-436 cells. SK-BR-3 cell was grown in McCoy’s 5A medium (PM150710). All media for BC cells were supplemented with 10% fetal bovine serum (164210) and 1% penicillin−streptomycin.
All cells were finally incubated in the Sanyo MCO-18AIC(UV) CO₂ incubator (SA-MC018, Marshall Scientific, LLC., Hampton, NH, USA) at 37°C and 5% CO₂. As TMTC3 was lowly expressed in MCF-7 and MDA-MB-231 cells, these two cells were used for subsequent studies.

The foreskin samples used in this study were donated by healthy adults aged 20–30 years who underwent surgical circumcision, and all donors were of the Asian race. Fresh foreskins were obtained with the donors’ consent and the approval of the Ethics Committee of the Fourth Medical Center of Chinese PLA General Hospital, Beijing, China. The foreskin tissue was surgically removed using aseptic techniques. The collected samples were kept in sterile PBS and transported at 2–8 °C to the laboratory for processing. To obtain the EpiSCs, subcutaneous fat was removed from skin samples with a scalpel, and skins were placed dermis side down in 2.4 U/mL Dispase II for 1 h. Skin was scraped to separate the epidermis from the dermis. Single-cell suspensions were obtained by trypsin digestion at 37 °C. Cells were then filtered through 70 mm, followed by 40 mm strainers, and plated on collagen IV-coated 6-well tissue culture dishes to establish primary cell lines as described [10 (link)]. Independent clones were cultured and passaged in Epilife culture medium (MEPI500CA, Gibco) with Human Keratinocyte Growth Supplement (HKGS) (S0015, Gibco). The 2nd and 3rd passages were used in the experiments. For experiments, cells were cultured in Epilife culture medium with or without Erlotinib (2 μM, HY-50896, MCE)), Tideglusib (200 nM, HY-14872, MCE) or EGF (20 ng/mL, P00033, Solarbio).