Dmem f12 medium

Goat mammary epithelial cells (GMECs) were isolated from mammary gland tissue, and purified as previously reported [10 ]. These cells were treated in a lactogenic medium for 48 h to induce differentiation into secretary cells [11 (link), 12 (link)]. For cell experiments, GMECs were cultured in DMEM/F12 medium (Hyclone Laboratories, Beijing, China), containing 5 μg/mL insulin, 5 μg/mL hydrocortisone, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 ng/mL epidermal growth factor 1 (EGF-1, Gibco, Gaithersburg, MD, USA), and 10% fetal bovine serum (FBS, Biological Industries, BeitHaemek, Israel) in a humidified incubator with 5% CO₂ at 37 °C. Synthetic miRNA mimics were purchased from RiboBio (Guangzhou, Guangdong, China) and transfected into GMECs using the K2 transfection system (Biontex Laboratories GmbH, München, Germany) according to manufacturer’s instructions.

The umbilical cords were taken from a healthy fetus delivered by a maternity cesarean section of the obstetrics department of Henan Provincial People’s Hospital. It was treated within 1 h after aseptic collection. Infectious diseases such as hepatitis B, hepatitis C, syphilis, AIDS, cytomegalovirus and Epstein-Barr virus are excluded by serological testing before maternal surgery. Written informed consent was obtained from the donors. Fresh umbilical cords were washed with 0.01 M pH 7.2~7.4 phosphate buffer saline (PBS) supplemented with antibiotics (100 U/ml of streptomycin, 100 U/ml of penicillin) twice to remove blood. After treated with 70%ethanol, umbilical cords were then minced into small pieces and incubated with DMEM/F12 medium (1:1) (Hyclone Laboratories; Thermo Fisher Scientific Life Sciences, USA) supplemented with 10%fetal bovine serum (FBS; Hyclone Laboratories) in dishes at 37 °C, 5%CO₂ supplement. Cells were trypsinized and collected for subculture when they reached 80% confluence. Only uMSCs in passages 2–5 were used in our study. Cultured cells were fluorescence marked with specific mensenchymal stem cell surface antigen CD29, CD44, CD34 and CD45 (BD biosciences, New Jersey, US) and identified by flow cytometry.

The human gastric epithelial AGS cell line (derived from a human gastric adenocarcinoma) was cultured in a DMEM/F12 medium (HyClone Laboratories Inc., Logan, UT, United States), with supplementation of 10% FBS (PANS, Aidenbach, Bayern, Germany) at 37°C in a 5% CO₂ humidified atmosphere. For H. pylori infection assays, AGS cells were grown in 6-well plates (NUNC, Thermo, DE, United States) until the confluence reached 75% in DMEM/F12 medium containing 10% FBS. Before infection, the supernatant was removed, and cells were washed twice with phosphate-buffered saline (PBS), followed by culture in FBS-free DMEM/F12 for 4 h. H. pylori strains were first cultivated on agar plates; then, the bacteria were collected and resuspended in Brucella broth at an initial OD₆₀₀ of 0.1, followed by culture for 24 h. Bacterial cells were then pelleted and washed twice with DMEM/F12 medium, resuspended in DMEM/F12 medium, and added to the AGS cell culture at a multiplicity of infection (MOI) of 100.

Three cell lines were used for biological assays. The human colon adenocarcinoma cell line (HCT-116) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human breast adenocarcinoma cell line (MCF-7) was obtained from the collection of the Maria Skłodowska-Curie Memorial Cancer Center and the National Institute of Oncology branch in Gliwice, Poland. The Normal Human Dermal Fibroblasts-Neonatal (NHDF-Neo) was purchased from Lonza (Cat. No. CC-2509, NHDF-Neo, Dermal Fibroblasts, Neonatal, Lonza, Warsaw, Poland). All cells were cultured as a monolayer in a complete medium under standard conditions in a humidified incubator with 5% CO₂ at 37 °C. The complete culture media consisted of RPMI 1640 or DMEM/F12 medium (HyClone Laboratories, Inc., Logan, UT, USA), supplemented with 10% of heat-inactivated fetal bovine serum (FBS; EURx, Gdańsk, Poland) and 1% of Antibiotic Antimycotic Solution: penicillin (10,000 U/mL) and streptomycin (10,000 µg/mL) (Sigma-Aldrich, Taufkirchen, Germany).

Human renal cancer 786-O and ACHN cell lines were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in RPMI-1640 medium (Cytiva, SH30011.02) and MEM medium (Cytiva, SH30193.04). The human proximal tubule epithelial HK2 cell line was incubated with DMEM/F12 medium (Cytiva SH30004.03). These cell culture media contained 10% FBS (Cytiva SH30071.02), NEAA solution (100X; Cytiva SH30238.01), P/S reagent (100X, Cytiva SV30010) and cells incubated at 37 °C (involved in 5% CO₂) in a humidified atmosphere. RCC and HK-2 cells were subcultured when cells reached 70% confluence.

The Neuro 2A (N2a) cell line was obtained from the American Type Culture Collection (ATCC, #CCL-131). N2a cells were cultured in DMEM/F12 medium (Cytiva) containing 10% FCS, 15 mM HEPES, 2.5 mM L-glutamine and 0.1 mg/ml penicillin and streptomycin. Transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). In brief, N2a cells were seeded at 1 × 10⁵ cells/well in 24-well plates, and transfected 24 h later with the indicated DNA plasmid (0.5 μg/well) using Lipofectamine 2000 transfection reagent (1 μl/well). pcDNA FRT TO- APP695 and pcDNA FRT TO- APPSwed/Ind were gifts from Aleksandra Radenovic (Addgene plasmids #114193 and #114194, respectively). pcDNA3.3 plasmid (Themo Fisher Science, #K830001) was obtained from Invitrogen, and was used as a negative control (Empty).