Dmem f12 medium

The endometrial tissues collected from patients with ovarian endometriosis were washed twice with PBS, cut into approximately 5 mm³ pieces, and digested with 2.5 mg/ml type IV collagenase (Sigma-Aldrich, USA) for 1 h at 37°C and 15 U/ml deoxyribonuclease (Sangon Biotech, China) for 30 min. ESCs in the cell suspension were purified according to the following procedure: filtered through a 70 μm cell strainer, filtered through a 50 μm cell strainer, and washed twice with PBS. ESCs were cultured in DMEM/F12 medium (Procell, China) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), and maintained at 37°C with 5% CO₂. ESCs at passage two were used for immunofluorescence staining for detection of vimentin and cytokeratin. ESCs with purity greater than 90% were used in subsequent studies.
Recombinant human TGF-β1 (Sangon Biotech, China) at concentrations of 0–10 ng/ml was used to treat ESCs for 24 h to induce EMT [32 (link),33 (link)]. The ERK1/2 inhibitor PD98059 (MedChemExpress, USA) at a density of 30 μM was used to treat the cells for 24 h.

Ninety-six tumor samples and their adjacent normal tissues (2 cm from the lesion) were obtained from patients with primary NSCLC in the Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, between March 2014 and September 2016. None of the patients had received radiotherapy or chemotherapy before the surgical operation. The study was approved by the Ethics Committee of Sichuan Cancer Hospital & Institute, Sichuan Cancer Center and was conducted following the Declaration of Helsinki (2013 revision). The patients provided informed written consent. Samples were kept in liquid nitrogen for analysis.
The human NSCLC cell lines A549, NCI-H1299, HCC827, NCI-1650, and NCI-H358 were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China), and the normal human lung epithelial cell line DEAS-2B was obtained from Beyotime (Shanghai, China). A549 cells were maintained in DMEM/F12 medium (Procell, Wuhan, China) with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). The remaining cell lines were cultured in RPMI 1640 medium (Procell, Wuhan, China) containing 1% Glutamax (Invitrogen), 1% (v/v) 100 mM sodium pyruvate solution (Invitrogen, Waltham, MA, USA), and 10% FBS. All cells were grown in a humidified 37 °C incubator with 5% CO₂.

A total of 66 pairs of BC tissue samples and adjacent normal tissue samples were obtained from BC patients at the Nanyang Second General Hospital, and all tissues were stored at -80℃. The clinical characteristics of these patients were shown in Table 1. All patients had not undergone any chemotherapy and radiotherapy before surgery. The experiment here got approval from the Ethics Committee of Nanyang Second General Hospital.Table 1

Clinical characteristics of patients with breast cancer (n = 66)

Characteristic	Cases
Age
< 50	38
≥ 50	28
Histologic grade
I	3
II	45
III	18
Lymph node metastasis
Yes	35
No	31
T stage
T1/T2	52
T3/T4	14
N stage
N0/N1	39
N2/N3	27

BC cell lines (MDA-MB-231 and MCF-7), human embryonic kidney cell line 293 T and human normal mammary epithelial cell line (MCF10A) were obtained from Procell (Wuhan, China). MDA-MB-231 cells were maintained in Leibovitz's L-15 medium (Procell); MCF-7 cells were maintained in Minimum Essential Medium (MEM) medium (Procell); MCF10A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Procell); 293 T cells were cultured in DMEM medium (Procell). All mediums were added with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Rockville, MD, USA) and 1% penicillin–streptomycin liquid (Gibco). All cells were cultured at 37℃ at 5% CO₂.