Mcf 7

Formaldehyde-fixed paraffin-embedded (FFPE) BC tissues and unpaired mammary hyperplasia (non-tumor tissues) were randomly collected from patients who had undergone surgery at the Shaanxi Provincial People's Hospital in China. Clinicopathological data such as age and gender, as well as histological data, tumor size, lymph node metastasis status, ER status, PR status, and AR status were obtained by reviewing their pathology records. Specimens were collected after obtaining written informed consent from the patients as well as approval of the ethical committees. Patient anonymity was maintained throughout the study. Human BC cell lines MCF-7, HCC1937, MDA-MB-231 and MDA-MB-435, human breast epithelium cells HBL-100 [23 (link)] and human embryonic kidney (HEK) 293T cells were obtained from the Cell Bank (Shanghai Institute of Biochemistry and Cell Biology, CAS, Shanghai, China).Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Biological Industries) and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin sulfate). Cells were grown in 5% CO₂ at 37 °C. The cell line was tested for mycoplasma contamination using the Mycoplasma Detection Kit (Beyotime, Haimen, China) and was found to be negative.

MCF10A, HCC1937, MDA‐MB‐231, MCF7, T47D, BT474, and SKBR3 cell lines were purchased from the ATCC. The HCC1937, MDA‐MB‐231, MCF7, T47D, BT474, and SKBR3 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, 06‐1055‐57‐1ACS; Biological Industries) with 10% fetal bovine serum (FBS, 04‐001‐1ACS; Biological Industries) and 100 U/mL penicillin–streptomycin. MCF10A cells were maintained in DMEM/F12 (1:1) containing 5% horse serum, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 20 ng/mL EGF, 0.1 μg/mL cholera toxin, and 100 U/mL penicillin–streptomycin. The MDA‐MB‐231 cells were treated with SGC707 (MCE, HY‐19715) at concentrations of 4 and 8 μmol/L.

The human breast cancer MDA-MB-231, MCF-7 and SK-BR-3 cell lines, and the human mammary epithelial MCF-10A cell line, were purchased from the Shanghai Institute of Biochemistry and Cell Biology. MDA-MB-231 and MCF-7 cells were cultured in complete DMEM supplemented with 10% FBS (both Biological Industries), 100 U/ml penicillin and 100 µg/ml streptomycin. SK-BR-3 and MCF-10A cells were cultured in RPMI-1640 (Biological Industries) containing 1.5 mg/ml NaHCO₃, 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. All cell lines were cultured at 37°C in a 5% CO₂ atmosphere.
Short hairpin RNA (shRNA) targeting AQP1 mRNA (sh-AQP1; 5′-CCATTATGCTGGTGTATGT-3′) (GV248-AQP1) and the corresponding negative control shRNA with a non-targeting AQP1 sequence (sh-NC; 5′-TTCTCCGAACGTGTCACGT-3′) (GV248-NC) were designed and synthesized by Shanghai GeneChem Co., Ltd., and the concentrations were both adjusted to 1×10⁸ TU/ml. The sh-AQP1 and sh-NC (MOI=20) were transfected into MDA-MB-231 cells (3.0×10³ cells per well) using polybrene (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The duration of transfection was 12 h at 37°C followed by changing the fresh medium. Transfected cells were used for subsequent experiments after 72 h.