Fig 2: MTDH expression is associated with stem cell like property in metastatic PDAC cells and correlates with anoikis resistance with epithelial property in KPCY cells(A) MTDH protein expression in human pancreatic cell lines was analyzed by western blot. Metastatic PDAC cell lines (CFPAC-1 and Hs766T) showed higher levels of MTDH compared to primary invasive PDAC cell lines (Capan-2, PANC-1, and MIA Paca-2). (B) MTDH knockdown in PANC-1 and CFPAC-1 cells by MTDH siRNA-1 and -2 was confirmed by western blot analysis. (C) The sphere formation rate in PANC-1 and CFPAC-1 cells treated with negative control siRNA, MTDH siRNA-1 or -2. Upper panel: Representative sphere forming cells of CFPAC-1 cells in pancreatosphere formation assays. Sphere cells in CFPAC-1 cells treated with control siRNA (left panel), MTDH siRNA-1 (middle panel), and MTDH siRNA-2 (right panel). Lower panel: MTDH knockdown significantly decreased the sphere formation rate in CFPAC-1 cells, but not in PANC-1 cells (student’s t-test). (D) CD133 expression and EpCAM expression in CFPAC-1 were analyzed by flow cytometry. The proportions of CD133+ cells and EpCAMHigh cells were significantly decreased by MTDH knockdown in CFPAC-1 cells (student’s t-test). Values are shown as mean ± SEM. (E) MTDH knockdown by MTDH shRNA-1 and -2 was confirmed by western blot analysis. (F) Upper panel: Various morphological changes of KPCY cells in 3D organotypic cell culture. Lower panel: MTDH knockdown significantly decreases the ratio of spheroid cyst among KPCY cells treating with shRNAs. (G) The number of colonies was significantly reduced by MTDH knockdown in KPCY cells (student’s t-test).

Fig 3: MTDH knockdown decreased the incidence of liver metastases in experimental mouse PDAC models(A) Experimental design for orthotopic transplantation of KPCY cells transduced with control shRNA (n=14), MTDH shRNA-1 (n=14), or shRNA-2 (n=14). (B) Experimental design for intra-portal vein injection of KPCY cells transduced with control shRNA (n=17), MTDH shRNA-1 (n=16), or shRNA-2 (n=14). (C) Difference in the incidence of liver metastasis between orthotopic transplantation model (left panel) and intra-portal vein injection model (right panel). (D) The liver metastasis formation rate of KPCY cells transduced with either control shRNA, MTDH shRNA-1, or shRNA-2 in both orthotopic transplantation model and intra-portal vein injection model. MTDH knockdown significantly decreases the incidence of liver metastases in these two models. (E) Quadruple immunofluorescence staining for YFP (green), MTDH (white), E-cadherin (red) and DAPI (blue) in metastatic colonies of liver tissue. MTDH and E-cadherin are co-expressed in liver metastases of PDAC. Bar, 50µm.

Fig 4: AEG-1 is a direct target of miR-506 in human osteosarcoma MG63 cells. (A) Specific binding locations of miR-506 and the 3'-UTR of AEG-1. (B) Luciferase reporter assay showed that the relative luciferase activity of AEG-1 3'-UTR was significantly decreased in MG63 cells co-transfected with wt or mut AEG-1 3'-UTR and miR-506 mimics. (C and D) Western blot analysis showed that the relative level of AEG-1 protein (64 kDa) was decreased in MG63 cells following transfection with miR-506 mimics, but was increased in human normal osteoblastic hFOB 1.19 cells following transfection with anti-miR-506 mimics. *P<0.05, **P<0.01 vs. miR-control; #P<0.05 vs. anti-miR-control. miR, microRNA; AEG-1, astrocyte elevated gene-1; UTR, untranslated region; wt, wild-type; mut, mutant.

Fig 5: The interaction of estrogen receptor (ER) and metadherin (MTDH) in endometrial cancer and breast cancer cell lines. (A) Reciprocal co-IP analysis of the interaction between MTDH and ER in ECC1 cells. Cell lysates of ECC1 cells treated with 10 nM estrogen were immunoprecipitated with anti-MTDH or anti-ERa antibody. (B) Reciprocal co-IP analysis of the interaction between MTDH and ER in MCF-7 cells. Cell lysates of MCF-7 cells treated with 10 nM estrogen were immunoprecipitated with anti-MTDH or anti-ER antibody. (C) Reciprocal co-IP analysis of the fractions in ECC1 cells. Cytoplasmic and nuclear fractions of ECC1 cells treated with 10 nM estrogen were immunoprecipitated with anti-ERa antibody.