Mesenchymal Stem Cells and Osteosarcoma Cell Lines

Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated and cultured as previously described [23 (link)]. The human OS cell lines MG63, Saos2, HOS, and U2OS were purchased from the American Type Culture Collection (ATCC). BM-MSCs and human OS cell lines were cultured in DMEM (CR-12800, Cienry) supplemented with 10% FBS (11011-8611, Hangzhou Sijiqing Co., Ltd), penicillin (50 U/mL; Gibco), and streptomycin (50 µg/mL; Gibco), and cells were maintained in humidified 37 °C incubators with 5% CO₂. Antibodies against CCR9 (ab32556), total β-catenin (ab68183), active β-catenin (ab246504), and GAPDH (ab9485) were purchased from Abcam. Antibodies against E-cadherin (sc-8426), N-cadherin (sc-59987), vimentin (sc-6260), Twist (sc-81417), Snail (sc-271977), and MMP-1 (sc-21731) were obtained from Santa Cruz Biotechnology. Primary antibodies were used for western blotting at a dilution of 1:1000, and secondary antibodies were used at a dilution of 1:3000. The primary antibodies and the secondary antibodies for immunofluorescence were used at dilutions of 1:400 and 1:1000. The pathway inhibitor XAV-939 (HY-15147, Med Chem Express, Inc.) was used to inhibit the Wnt/β-catenin signaling pathway as previously described [24 (link)].