Anti dkk1 neutralizing antibody

The following drugs were used for cell interventions as described in each experiment: medroxyprogesterone acetate (MPA) (Sigma Aldrich, USA), DKK1 (Sino Biological, China), CHIR-99021 (Selleck, USA), anti-DKK1-neutralizing antibody (R&D System, USA), and Foscenvivint (Selleck, USA).

HUVECs were labeled with the lipophilic fluorescence tracer, dioctadecyloxacarbocyanine perchlorate (DiO). The 8-well chamber slides were coated with Matrigel (BD Biosciences), and approximately 2.5 ×10³ HUVECs/well were harvested immediately with Endothelial Cell Growth Basal Medium (Lonza Bioscience) supplemented with recombinant DKK-1 (100 ng/mL), control IgG (15 mg/mL), or anti-DKK-1 neutralizing antibody (15 mg/mL) (R&D Systems). Cells were cultured at 37 °C in 5% CO₂, and time-lapse images were captured for 48 h using a CSU-X1 spinning disk confocal (Yokogawa, Tokyo, Japan) and Andor iXon3 EMCCD camera system (Andor Technology, Belfast, UK). Images were analyzed using the iQ software (Andor Technology).