Thp 1

The human monocytic cell line THP-1 was purchased from ATCC (TIB-202) and maintained in at 37° C and 5% CO₂ in RPMI 1640 medium with 10% FBS. The THP-1 macrophages were obtained through treating the THP-1 monocytes with 100 ng/mL phorbol-12-myristate-13-acetate (PMA, MedChemExpress, Monmouth Junction, NJ, USA) for 48 h. Then the cells were transfected with pcDNA3.1 or pcDNA3.1-IGFBPL1 using GP-Transfect-Mate (Jima, Shanghai, China) according the to the instruction. After 24 h of transfection, THP-1 macrophages were treated for 72 h with different reagents.

OC cell lines SKOV3 (CL-0215) and A2780 (CL-0013), and a human monocytic leukemia cell line THP-1 (CL-0233), were acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China). OC cells were cultured in DMEM (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% fetal bovine saline (FBS) and 1% antibiotics in a humidified environment at 37° with 5% CO₂. Exponentially growing cells were used for subsequent experiments. THP-1 cells were incubated with 150 nM phorbol 12-myristate 13-acetate (PMA, ab120297, Abcam Inc., Cambridge, MA, USA) for 24 h and then cultured in complete RPMI-1640 for 24 h to differentiate into M0 macrophages. For SK treatment, every 1 × 10⁴ OC cells were treated with 5 µM of SK (HY-N0822, MedChemExpress, Monmouth Junction, NJ, USA) dissolved in dimethyl sulfoxide (DMSO) for 48 h. For exo (see preparation details later) treatment, every 1 × 10⁴ THP-1 cells were treated with 50 µg exo for 72 h. Cells without SK or exo treatment were set to controls. For β-catenin inhibition, THP-1 cells were treated with 10 µM of β-catenin/Tcf inhibitor FH535 (219330, Sigma-Aldrich, Merck KGaA, Darfmstadt, Germany) for 12 h, and those treated with an equal volume of DMSO solution were set to controls. All procedures were conducted in a sterile condition.