Fig. 5

eMSCs inhibited Wnt/β-catenin signaling in EC cells through DKK1. A, B eMSCs inhibited expression of β-catenin protein and Wnt target genes in EC organoids and cell lines. CM from eMSCs was used to treat EC organoids for 96 h or EC cell lines for 48 h. EC cells were then harvested for qRT-PCR and western blotting. EC-case 2 organoid line was used in Fig. 5A. C Expression of β-catenin protein was reduced in cytoplasm and nuclei of EC cells after treatment of CM derived from eMSCs. Original magnification, 40 × ; Scale bar, 80 μm. D DKK1 inhibited expression of β-catenin and Wnt target proteins (AXIN2 and C-MYC) in EC cell lines. EC cells were treated with DKK1 (100 ng/ml) for 48 h and then harvested for western blotting. E DKK1-neutralizing antibody compromised the inhibitory effect of eMSCs on Wnt signaling in EC cells. CM obtained from eMSCs was pre-treated with DKK1-neutralizing antibody (anti-DKK1) or isotype control (IgG) overnight and then used to treat EC cells for 48 h. EC cells were harvested for western blotting analysis. F Silencing DKK1 by siDKK1-3 rescued Wnt signaling, which was inhibited by eMSCs in EC cells. EC cells were treated with the indicated CM of eMSCs for 48 h. EC cells were harvested for western blotting analysis. NM, normal medium; CM, conditioned medium. The blots of AXIN2, C-MYC, β-catenin, and GAPDH were all cropped (B, D-F) and full-length blots were presented in Additional file 7: Figure S6. Data were analyzed by unpaired t-test (A). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001