Fig. 4

eMSCs inhibited EC cell proliferation and stemness through paracrine secretion of DKK1. A eMSCs showed the highest level of DKK1 mRNA, compared to AD-MSCs and UC-MSCs, while the mRNA levels of SFRP1 and WIF1 were not significantly elevated in eMSCs, compared to other MSCs. B eMSCs showed the highest level of DKK1 in eMSCs cell, compared to AD-MSCs and UC-MSCs, as detected by western blotting. C eMSCs showed the highest level of DKK1 secretion compared to AD-MSCs and UC-MSCs. CM obtained from the three MSCs was collected under the same condition as shown in Materials and Methods and the DKK1 levels were evaluated using an ELISA kit. D-E DKK1-neutralizing antibody compromised the inhibitory effect of eMSCs on proliferation (D) and stemness (E) 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 Cell Viability Assay (D) and qRT-PCR (E) respectively. F–G. The mRNA and protein levels of DKK1 were reduced in eMSCs by transfection of DKK1 siRNAs. eMSCs were transfected with three different siRNAs (siDKK1-1, -2, and -3) targeting DKK1 or siMock for 6 h. At 48 h after transfection, eMSCs were harvested for examination of mRNA and protein levels of DKK1. siDKK1-3 was used for further investigation. H-I. Silencing DKK1 in eMSCs abrogated the anti-proliferation and anti-stemness effects of eMSCs on EC cells. The CM of eMSCs was collected after 24 h of siDKK1-3 transfection. EC cells were treated with the indicated CM of eMSCs for 48 h and then the mRNA levels of stemness-associated genes, ALDH1, BMI1, and NANOG, were measured by qRT-PCR. NM, normal medium; CM, conditioned medium; The blots of DKK1 and GAPDH were all cropped (B and G) and full-length blots were presented in Additional file 7: Figure S6. Data were analyzed by unpaired t-test (A, C, E, F, I) and ratio t-test (D and H). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001