Highgene

Cells were seeded into 6-well plates for transient knockdown transfection, followed by the transfection of 100pmol siRNA-FOXC1 (GenePharma, Shanghai, China) using HighGene (ABclonal, Wuhan, China) ECAh well, following the manufacturer’s guidance. Cells were seeded into 6-well plates for transient gene overexpression transfection, followed by the transfection of the plasmids expressing CBX7 or IGF-1R were purchased from Genechem (Shanghai, China) using HighGene (ABclonal, Wuhan, China).
At 48 h post-transfection, transfection efficiency was assessed using RT-qPCR and western blot. For stable transfection, ECA-109 and KYSE-150 cells were transfected with lentivirus vectors encoding either FOXC1-targeting shRNA or non-targeting control shRNA, following the manufacturer’s instructions (Genechem, Shanghai, China). Briefly, cells were seeded in 6-well plates, and when the cell density reached 30%, a medium containing viral fluid at an MOI of 10, without serum, was added. This medium was replaced with a complete medium 24 h later. After lentiviral infection, ECA-109 cells and KYSE-150 cells underwent a two-week selection process with 1 µg/mL puromycin to obtain stable clones. Transfection efficiency for each vector was evaluated through a western blot.

Human HCT15, RKO, HCT8, SW620, HCT116, HIEC-6, and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, CA, USA) at 37 °C in a 5% CO₂ incubator. For transfection, after growing to 70% confluence, cells were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) or HighGene (ABclonal, Wuhan, China), according to the manufacturer’s instructions.

GCs were isolated and cultured using previously described methods [12 (link)]. Briefly, fresh ovaries from adult sows were washed alternately with saline and 75% alcohol, and follicular fluid from healthy follicles was drawn, collected into centrifuge tubes, and centrifuged to collect cells. After washing with phosphate-buffered saline, the cells were resuspended using DMEM/F12 medium (containing 15% fetal bovine serum and 1% penicillin), seeded into cell culture plates, and incubated at 37 °C in a 5% CO₂ incubator. When both GC density and status reached the transfection requirement, plasmids (1000 ng per well) were transfected into GCs by using HighGene (ABclonal, Wuhan, China) according to instructions. After 24 h of transfection, GCs (5 × 10⁵) were collected for luciferase reporter assay. Luciferase activity was measured using a luciferase reporter assay kit (Vazyme, Nanjing, China) according to the instructions.