NDRG1 was fused with LexA DNA-binding domain at N- or C-terminal, and LRP6 was fused with B42 activation domain. Appropriate combinations of expression plasmids were transformed in the same yeast strain EGY48. The resultant transformants were grown and assayed for the β-galactosidase activity of the reporter LacZ gene. pBait and pTarget are a pair of positive controls. Inset image shows transformants grown in an agar plate containing X-gal.Co-IP analysis for the interaction of NDRG1 and LRP6 in mammalian cells. PC3mm/Tet cells were transfected with the expression plasmid of HA-LRP6, treated with tetracycline to induce NDRG1. Cell lysates were precipitated with anti-HA for LRP6 (lanes 3 and 4) or anti-Flag for NDRG1 (lane 5) followed by Protein-G agarose. Western blot was performed to detect NDRG1 and LRP6.PC3mm/Tet and MCF7/Tet cells were treated with (lanes 1 and 2) or without (lanes 3 and 4) tetracycline to induce NDRG1 in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Wnt. Cell lysates were subjected to Western blot with antibodies against phospo-LRP6, total-LRP6, Flag-NDRG1 and β-tubulin.Western blot analysis to confirm results from phospho-site profiling analysis for phospho-GSK3β (Tyr^279/216), GSK3β, phospho-Akt (Ser⁴⁷³) and Akt expression in PC3mm/Tet and MCF7/Tet cells that were treated with or without tetracycline.PC3mm/Tet and MCF7/Tet cells were transfected with the TOPflash reporter plasmid in the presence or absence of Wnt followed by treatment with tetracycline or not. Luciferase activity of cell lysates was assayed and normalized by internal Renilla luciferase activity. Values are from three independent experiments in triplicate. p values were calculated by two-sided Student's t-test.Western blot analysis of β-catenin expression in PC3mm/Tet cells treated with or without tetracycline prior to the addition of Wnt.PC3mm/Tet-Flag-NDRG1 cells were transfected with YFP-β-catenin and RFP expression plasmids in the presence or absence of tetracycline. After 48 h, the fluorescence intensity of β-catenin was determined under a confocal microscope. Each value was normalized by the RFP fluorescence intensity. Right panels show representative microscopic photos for each experimental group. Values are from three independent experiments in triplicate. p values were calculated by two-sided Student's t-test.

A. The expression of ATF3 was examined by qRT-PCR or ATF3 reporter assay in PC3mm (left panel) or MCF7 (right panel) that were transfected with or without the expression plasmid of β-catenin/TCF. Western blot was also performed to confirm the ATF3 expression in the presence of β-catenin/TCF. Values are from three independent experiments in triplicate.
B-C. Three-base mutation was introduced into the consensus sequence of β-catenin/TCF binding site on the ATF3 gene promoter of the CAT-reporter plasmid. Wild-type and mutant reporter plasmids were then transfected into PC3mm/Tet or MCF7/Tet cells in the presence or absence of tetracycline (B) or Wnt (C). CAT activity of the cell lysates was assayed and normalized by internal Renilla luciferase activity. Western blot analysis of ATF3 expression was also performed. Values are means ± SD of triplicate measurements.
D. ChIP assay for PC3mm/Tet cells treated with or without tetracycline for 48 h. Precipitated DNA was subjected to q-PCR using primers specific for the β-catenin binding consensus sequence on the ATF3 promoter (F1R1) as well as a control primer set (F2R2). The ratio of the precipitated DNA was calculated based on cyclic threshold value for each reaction. Values are means ± SD of triplicate measurements. For all experiment, p values were calculated by two-sided Student's t-test.

EMT-like morphological changes of PC3mm/Tet cells that were treated with or without Wnt in the presence or absence of tetracycline.Western blot analysis for E-cadherin and ATF3 expression (left panel) and immuno-fluorescence staining of β-catenin (right panel) in PC3mm/Tet cells that were treated with or without tetracycline prior to the addition of Wnt.PC3mm/Tet-Flag-NDRG1 cells were cultured in the presence or absence of Wnt with or without the treatment of tetracycline. RNA were then prepared and subjected to qRT-PCR analysis for the expression of Slug, Fibronectin, Vimentin and Twist. Values are from three independent experiments in triplicate. p values were calculated by two-sided Student's t-test. Western blot analysis was also performed to confirm the expression of NDRG1 (inserted fig).NDRG1 expression correlates with epithelial traits in a panel of prostate (left panel, GSE9633) and breast (right panel, Neve et al, 2006) cancer cell lines. Heatmap depicts the relative expression of normalized expression values of E-cadherin, NDRG1, Fibronectin1 and Vimentin for each cell lines. Green colour indicates low expression and red colour indicates high expression. Representative cell lines exhibiting epithelial-like and mesenchymal morphologies were marked with red and green colours, respectively.

A-C. PC3mm/Tet cells were treated with or without Wnt followed by treatment of tetracycline or not, and were subjected to assays for invasion (A), anoikis resistance (B) and adhesion assays (C).
D. PC3mm/Tet cells were transfected with the expression plasmids of β-catenin/TCF or vector control plasmids followed by treatment of tetracycline or not. Cells were then stained with CellTracker Green and injected into the lateral vein of nude mice (n = 3). Lungs were removed after 48 h and sectioned followed by counting the number of tumour cells under the confocal microscope. Lung vasculature was visualized by staining with rhodamine-conjugated lectin. For all experiment, values are from three independent experiments in triplicate. p values were calculated by two-sided Student's t-test.

To test tumorigenic and metastatic ability of WB1-1cell lines, 5 × 10⁵ cells of each line were injected into the mammary fat pad of nude mice. Non-invasive BLI was performed to monitor the primary tumour growth and the development of metastasis. At the time of necropsy, lungs and bones from individual mice were dissected and imaged ex vivo for bioluminescence. Shown are representative bioluminescent images of primary tumour and metastasis of individual mice (n ≥ 9).Summary of the tumour size and metastatic incidence at day 43.H&E-stained sections of mouse lungs from each group at the endpoint (left panel). The scale bar represents 100 µm. Metastatic growth of tumours in the lungs was also measured by BLI after necropsy (right panel). Data are presented as the mean photon flux. Error bars represent the SEM. p values were calculated by two-sided Student's t-test.Incidence of hind limb bone metastasis was represented by Kaplan–Meier curves, and their metastatic growth was measured by BLI. p values were calculated by Log-rank test and two-sided Student's t-test.Immuno-fluoresence staining of β-catenin and ATF3 in mouse primary tumours. The percentage of ATF3 positive tumour cells were quantified for each group. p values were calculated by two-sided Student's t-test.

Paraffin block sections of prostate (n = 29) and breast (n = 33) cancer specimens from patients were subjected to IHC analysis using antibodies to NDRG1 and β-catenin. Upper and lower panels show normal grade and high-grade tumours, respectively. Right panel; correlations of NDRG1 and β-catenin expression were calculated by the χ² test.IHC analysis was performed for the same set of prostate cancer specimens to determine the association of ATF3 with NDRG1and β-catenin as well as the patient metastatic status.Expression of NDRG1 and Wnt-related factors in prostate cancer patients were determined using the Lapointe cohort (GSE3933). p values were calculated by Mann–Whitney t-test.Kaplan–Meier survival analysis to determine the diagnostic value of the combined Wnt, LRP6 and NDRG1 in the database of Yu cohort (GSE6919). The relevance to relapse-free survival of patients was assessed by calculating relative hazard ratio and log rank p-value.

A-B. WB1-1 cells with or without shRNA for NDRG1 were treated with Dp44mT or vehicle alone. NDRG1 mRNA expression was measured by qRT-PCR (A), and the motility and invasive abilities of tumour cells were also examined (B). Values are from three independent experiments in triplicate. p values were calculated by two-sided Student's t-test.
C. 231-BoM cells (5 × 10⁴) were inoculated into SCID mice by i.c. injection, followed by i.v. administration of Dp44mT (0.4 mg/kg) (n = 8) or saline (n = 10) for four consecutive days. The degree of clearance of the tumour cells in the mice was measured by BLI at day 9. Right panels show representative images of BLI at day 0 and 9. p values were calculated by two-sided Student's t-test.
D. 231BoM cells (1 × 10⁵) were inoculated into nude mice through intracardiac injection followed by the treatment with Dp44mT (0.4 mg/kg) (n = 10) or saline (n = 12) for five consecutive days, and the treatment was repeated 4 times with 2 days interval. The left panel indicates metastasis-free survival of the hind limb of mice. p values are based on Log-rank test. Middle and right panels: 231BoM/shNDRG1 and 231BoM/shScramble cells (1 × 10⁵) were inoculated into nude mice through intracardiac injection followed by the Dp44mT treatment (0.4 mg/kg, n = 12) as above. The middle panel shows the strength of BLI signal of bone metastasis. Data are presented as the mean photon flux, error bars represent the SEM. p values were calculated by two-sided Student's t-test. The right panel indicates metastasis-free survival of mice. p values were calculated by Log-rank test.