Sirna mate reagent

Breast cancer cell lines BT549 and T47D were maintained in RPMI-1640 medium with 10% fetal bovine serum. Breast cancer cell lines MDA-MB-231, MDA-MB-157, MDA-MB-453, MCF7 and BT474 were cultured using Dulbecco's Modified Eagle Medium containing 10% FBS. Penicillin/streptomycin were added to cell culture medium. All cell lines were obtained from ATCC. Stable cell clones were constructed by selection using puromycin (1 μg/mL). For siRNA transfection, cells were seeded about 1 × 10⁵ cells per well in medium with 10% FBS and transfected with siRNA using siRNA-mate reagent (GenePharma) after reaching 70% confluency.

HepG2 cells and LO2 cells were donated by the Beijing Institute of Basic Medical Sciences; they were cultured in DMEM (Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), and were placed in a 37 °C, 5% CO₂ incubator. The transfection of HepG2 cell was performed, utilizing si-ZNF385A, si-ZNF346, or si-NC (GenePharma, Shanghai, China), using siRNA-Mate reagent (GenePharma, China) following the manufacturer’s recommendations. The sequences for si-ZNF385A were sense (5′–3′) GCCCGUCAUUUCCUGUAAUTT and antisense (5′–3′) AUUACAGGAAAUGACGGGCTT. The sequences for si-ZNF346 were sense (5′–3′) GGGAAACGAAGAAGCUAGATT and antisense (5′–3′) UCUAGCUUCUUCGUUUCCCTT.

Three pairs of siRNA oligonucleotides targeting FOXO4 were synthesized by GenePharma Co., Ltd. The GAPDH sequences were used as a positive control. An unrelated sequence was used as a negative control (provided by GenePharma). The sequences were as follows: FOXO4 siRNA oligo-1: 5′-CGCGAUCAUAGACCUAGAUTTAUCUAGGUCUAUGAUCGCGTT-3′ (sense); FOXO4 siRNA oligo-2: 5′-CAGCUUCAGUCAGCAGUUATTUAACUGCUGACUGAAGCUGTT-3′ (sense); FOXO4 siRNA oligo-3: 5′-GUGACAUGGAUAACAUCAUTTAUGAUGUUAUCCAUGUCACTT-3′ (sense); GAPDH siRNA oligo (positive control): 5′-GUAUGACAACAGCCUCAAGTT-3′ (sense); and negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense).
According to the manufacturers’ instructions, FOXO4 siRNA oligos were transfected into cells using the siRNA-Mate™ reagent (GenePharma Ltd., Shanghai, China). After cultured for 2 to 3 days, total RNA and protein were extracted. For stable transfection, a lentiviral overexpression vector (Lenti-FOXO4) was constructed (Shanghai GeneChem Co., Ltd., Shanghai, China). Using a GV166-puro Vector (GeneChem Co., Ltd., Shanghai, China), a lentiviral vector that expressed GFP alone (LV-control) was used as a negative control (NC).