Ro4929097

HUVECs and human osteosarcoma cell lines (U2OS and 143B) were purchased from China Centre for Type Culture Collection (CCTCC; Wuhan, China). These cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Camarillo, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Cells were cultured in a constant temperature incubator at 37°C and 5% CO₂.
HUVECs were incubated with 10 μM GW4869 (exosome inhibitor; Sigma-Aldrich, St. Louis, MO, USA) or 0.1% DMSO for 24 h. GW4869 is a cell-permeable, noncompetitive N-Smase (neutral sphingomyelinase) inhibitor. GW4869 blocks the ceramide-mediated sprouting of multivesicular bodies, thereby inhibiting the release of exosomes from multivesicular bodies. Thus, GW4869 is a commonly used as an exosome inhibitor [15 ]. For co-culture, U2OS and 143B cells were incubated with the HUVEC supernatant, HUVEC-derived exosomes (VECs-Exo) or PBS. U2OS and 143B cells were treated with 1 mM RO4929097 (γ secretase inhibitor; Sigma-Aldrich) for 24 h. The Notch receptor is cleaved by furin convertase, ADAM and γ-secretase to produce Notch intracellular domain. The Notch intracellular domain is released to the nucleus and then activates the transcription of related genes [16 (link)]. Thus, RO4929097 blocks the Notch signaling pathway by inhibiting the activity of γ secretase.

Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primary antibodies were obtained from Abcam (Cambridge, MA USA), which included anti-Notch2, anti-Hey1, anti-Snail, anti-N-cadherin, anti-Twist, anti-E-cadherin, and anti-GAPDH antibodies. RO4929097 and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan) and all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.