Fig 2: Eudesmin treatment promotes the expression of pluripotency marker genes. (A) Gene expression values of representative cluster 1 genes (TET1, NCAN, COCH, and NOTCH3) in Oct4-depleted embryonic stem cells (GDS1824). (B) Gene expression values of representative cluster 2 genes (DKK1, ROR2, MBP, and HOXC8) in Oct4-depleted embryonic stem cells (GDS1824). (C) Immunoblot analysis of H7 embryonic stem cells treated with or without eudesmin (40, 80 µM) for 24 h. (D) The mRNA levels of NANOG, OCT4, and SOX2 genes in H7 embryonic stem cells treated with or without eudesmin (80 µM) for 24 h. Data represent means ± SEM for n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Treatment of WT control NPCs with recombinant Dkk1 mimics the phenotypes observed in nGD NPCs. (A) Western blot showing the expression of phosphorylated LRP6 (pLRP6) and (non-phospho) active ß-catenin in control (WT) NPCs that were either left untreated (NT) or were treated with 100 ng/mL rDkk1 (+rDkk1) for 24 h. The proteins were normalized to ß-actin, and fold-change was calculated with respect to NT WT. (B) Immunofluorescence staining of pGSK3ß (Ser9) (green) and the lysosomal marker LAMP1 (red) in control NPCs that were either left untreated (NT, top panel) or were treated with rDkk1 (+rDkk1, bottom panel), as in A. The nuclei are labeled with DAPI (blue). Scale bar: 50 µm. The fold-change in mean fluorescence intensity (MFI) is plotted below the images. (C) Representative Western blot showing LAMP1 expression in control NPCs treated as in A. The bands were normalized to ß-actin, and the plot below the WB shows fold-change. (D) Control cells were either left untreated (NT, top panel) or were treated with rDkk1 (+rDkk1, bottom panel). Untreated and treated cells were stained with Lysotracker (red) and imaged. Scale bar: 50 µm. The fold-change in Lysotracker count is plotted below the images. * p < 0.05 and ** p < 0.01 (n = 3, mean ± S.E.M, unpaired Student’s t-test).

Fig 4: Oncogenic validation of BM1 and BM2. A,B: Cell proliferation assays by CCK8 (A) and Colony formation (B) indicated the increased ability to proliferate of BM1 and BM2, compared to MDA-MB-231 cells. C: Quantitative analysis of B. D: Transwell assay demonstrated the increased ability to invade of BM1 and BM2, compared to MDA-MB-231 cells. E: Quantitative analysis of D. F: Wound healing assay demonstrated the increased ability to migrate of BM1 and BM2, compared to MDA-MB-231 cells. G: Quantitative analysis of F. H: Western blot demonstrated the higher expression levels of EMT markers including Snail1, Slug, ZEB1, ZEB2, Twist, Vimentin, and DKK1 in both BM1 and BM2, compared to MDA-MB-231 cells. ß-actin and GAPDH served as loading control. Values are equal to mean ± SEM. *p<0.05, **p<0.01.

Fig 5: Relaxin inhibits DKK1 expression in isolated cardiomyocytes. Relaxin treatment for 2-days significantly reduced expression of DKK1 in isolated cardiomyocytes compared to control cells (n = 14 cells/group). *Indicates p < 0.05. White lines = 25 µm.