Complete dmem

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.

Human HCC HepG2 cells and L02 hepatocytes were purchased from ATCC and Beijing Union Medical College Hospital, respectively. The use of L02 cell line was approved by the Institutional Ethics Committee at Nankai University. HepG2 cells and L02 cells were cultured in complete DMEM (Biological Industries [BI], Israel) and complete Roswell Park Memorial Institute 1640 medium (BI), respectively, containing 10% (v/v) FBS (BI) and 1% penicillin–streptomycin (Thermo Scientific, Waltham, MA, USA). All cells were incubated at 37°C with 5% CO₂.