MAD2B interacts with TCF4 in yeast and mammalian cells. A, TCF4 interacts with MAD2B in yeast. Yeast two-hybrid assay results for Y190 yeast cells that were co-transformed with pACT2-MAD2B and pAS2–1 containing full-length TCF4 (amino acids 1–596) or TCF4 derivatives (amino acids 1–200, 201–395, 396–596). Binding activity was scored: + for interaction, and – for no interaction. ++ stands for stronger interaction. B, TCF4 interacts with MAD2B in mammalian cells. HA-tagged TCF4 and FLAG-tagged MAD2B or FLAG-tagged MAD2 plasmids were co-transfected into HEK293T cells for 48 h. Cell lysate was collected and immunoprecipitated (IP) with anti-HA followed by Western blot (WB) analysis with an antibody (Ab) against FLAG or HA. C, cells co-transfected with HA-TCF4 and FLAG-MAD2B as in B were collected and immunoprecipitated with anti-FLAG followed by Western blot analysis with an antibody against FLAG or HA. D, MAD2B interacts with endogenous TCF4 in colon cancer cells. Whole-cell lysate was collected from HCT116 colon cancer cells and subjected to co-immunoprecipitation with anti-MAD2B followed by Western blot analysis with an antibody against TCF4 or MAD2B. E, MAD2B interacts with TCF4 in vitro. His-TCF4 was purified from HEK293T cells. His-TCF4 was incubated with GST-MAD2B or GST purified from bacteria. The MAD2B-TCF4 protein complex was immunoblotted with anti-TCF4. The top panel shows the binding of TCF4 to GST-MAD2B. An input and pulldown products were separated by SDS-PAGE and detected by Western blot analysis with antibodies specific for TCF4. The bottom panel is Coomassie Blue-stained gel showing relative amounts of GST and GST-MAD2B. F, MAD2B and TCF4 both localize in the nucleus. Plasmids expressing EGFP-MAD2B and ERFP-TCF4 fluorescent fusion proteins were co-transfected into HEK293T cells. Fluorescence images were analyzed by confocal microscopy. G, overexpression of stabilized β-catenin does not affect the interaction between MAD2B and TCF4. HA-tagged TCF4 and FLAG-tagged MAD2B were co-transfected into HEK293T together with FLAG-tagged β-catenin (β-CTN) or empty pRK5-FLAG vector. 48 h after transfection cell lysate was collected and immunoprecipitated with anti-HA followed by Western blot analysis with an anti-FLAG antibody.

MAD2B represses the transcriptional activity of TCF4. A, MAD2B represses the transcriptional activity of TCF4 in HEK293T cells. HEK293T cells in 6-well plates were co-transfected with 0.5 μg of TCF4, 0.5 μg of β-catenin, and/or 2 μg of MAD2B (or 2 μg of MAD2) plasmids as indicated. TOPFLASH (TOP) or FOPFLASH (FOP) plasmids (0.5 μg) were used for the reporter assay. Cell lysate was collected 48 h after transfection, and relative luciferase activity was measured according to a standard dual-luciferase assay protocol. The results are presented as the TOP/FOP ratio. B, MAD2B represses the transcriptional activity of TCF4 in HCT116 colon cancer cells. HCT116 cells in 6-well plates were transfected with 0.5 μg of TOPFLASH or FOPFLASH plasmid together with 2 μg of MAD2B (M2B) plasmid as indicated. Relative luciferase activity was measured. C, silencing MAD2B in HCT116 cells releases the repression of TCF4 transcriptional activity. TOPFLASH or FOPFLASH plasmid (0.5 μg) was transfected into HCT116-scramble (HCT-sc) and HCT116-siMAD2B (HCT-siM2B) cells followed by the dual-luciferase assay. pRL-TK (50 ng) was co-transfected in the above experiments as an internal control. Data represent the mean ± S.D. of triplicate determinations. *, p < 0.05 compared with control; **, p < 0.01 compared with control.

MAD2B suppresses TCF4-mediated transcription by inhibiting the DNA binding activity of TCF4. A, MAD2B suppresses TCF4-VP16-mediated transcription. TOPFLASH or FOPFLASH (0.2 μg) were co-transfected into HEK293T cells in 24-well plates with 0.2 μg of TCF4-VP16AD and/or 0.4 μg of MAD2B (or 0.4 μg of MAD2) plasmids as indicated. Relative luciferase activity was determined 48 h after transfection and is shown as the TOP/FOP ratio. pRL-TK (10 ng) was cotransfected as an internal control. Data represent the mean ± S.D. of triplicate determinations. **, p < 0.01 compared with control. B, silencing of MAD2B does not interfere with TCF4-β-catenin complex formation. SW480-scramble (SW-sc) and SW480-siMAD2B (SW-siM2B) cells were lysed 48 h after plating onto a 100-mm culture dish. Whole cell lysate was collected and immunoprecipitated (IP) with anti-TCF4 followed by Western blot analysis with an anti-β-catenin, anti-TCF4, and anti-MAD2B antibody. C, the middle region of TCF4 interacts with MAD2B. Functional domains of TCF4 and the corresponding deletion mutants are shown. HA-TCF4 derivatives expressing plasmids (amino acids 1–200, 201–395, or 396–596) were co-transfected with a FLAG-MAD2B plasmid into HEK293T cells. Whole cell lysate was collected 48 h after transfection and immunoprecipitated with anti-HA followed by Western blot (WB) analysis with anti-FLAG or anti-HA. CTNNB1, catenin-binding site; HMG, high mobility group. D, MAD2B blocks the DNA binding activity of TCF4. His-tagged TCF4 protein was purified from HEK293T cells transfected with the His-TCF4-expressing plasmid. His-tagged MAD2B and His-tagged MAD2 proteins were extracted from E. coli (BL21DE3). Electrophoretic mobility shift assay experiments were performed by incubating a ³²P-labeled TBS probe with purified His-TCF4, His-MAD2B, His-MAD2, bovine serum albumin (BSA), a cold specific TBS, and/or nonspecific probe as indicated. The band shift is indicated by the arrowhead.

MAD2B silencing induces fibroblastoid morphology and reduces E-cadherin expression. A, knockdown of MAD2B induces fibroblastoid morphology. SW480 colon cancer cells were infected with lentiviral short hairpin RNA pLKO.1-siMAD2B to knock down MAD2B or with control vector (pLKO.1-scramble). Cells were pooled 48 h after puromycin selection, and 8 × 10⁵ cells were re-plated onto 100-mm dishes. Photographs were taken with a phase-contrast microscope 72 h after seeding. SW480 cells infected with pLKO.1-siMAD2B and pLKO.1-scramble were further applied to the TOPFLASH reporter assay as described in Fig. 2C. **, p < 0.01 compared with control. B, knockdown of MAD2B (siM2B) reduces E-cadherin protein expression. Whole-cell lysate from cells as in A was collected and subjected to Western blot analysis with antibodies against E-cadherin and MAD2B. C, knockdown of MAD2B reduces E-cadherin mRNA expression. Cells as in A were treated with Trizol. Total RNA was harvested and used for quantitative real-time PCR with E-cadherin and MAD2B primers. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The inset graph showed that MAD2B mRNA level was knocked down in SW480 cells infected with pLKO.1-siMAD2B. Data represent the mean ± S.D., n = 3. *, p < 0.05 compared with control. D, MAD2B silencing down-regulates E-cadherin transcription. pGL3-cdh1-promoter reporter plasmid (0.25 μg) was transfected into HCT116-scramble and HCT116-siMAD2B cells in 6-well plates, and the relative luciferase activity was examined. pRL-TK (50 ng) was used as an internal control. Results are presented as the ratio of pGL3-cdh1 to pGL3-basic. Data represent the mean ± S.D., n = 3. *, p < 0.05 compared with control. E, MAD2B silencing in SW480 cells induces expression of the mesenchymal markers N-cadherin and vimentin. Two MAD2B-silenced SW480 cells (SW-siM2B and SW-siM2B-2) and SW480-scramble cells (1 × 10⁶) were seeded onto 100-mm culture dishes, and cell lysate was collected 36 h later and used for Western blot analysis with antibodies against E-cadherin, N-cadherin, vimentin, MAD2B, and γ-tubulin. F, MAD2B silencing in SW480 cells attenuates E-cadherin expression and induces F-actin redistribution. Cells as in A were subjected to immunofluorescence staining. SW480-scramble and SW480-siMAD2B cells were stained for E-cadherin with mouse anti-E-cadherin (1:50) followed by fluorescein isothiocyanate-conjugated anti-mouse IgG (1:100). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). F-actin staining was performed with Alexa-555-phalloidin (1:40).

MAD2B silencing induces Slug expression and promotes the Slug promoter binding activity of TCF4. A, MAD2B silencing induces Slug protein expression. 1 × 10⁶ of SW480-scramble and SW480-siMAD2B cells (SW-siM2B and SW-siM2B-2) were seeded onto 100-mm culture dishes, and total cell lysate was collected 36 h later and subjected to Western blot analysis with antibodies against Slug and MAD2B. B, MAD2B silencing induces Slug mRNA expression. Total RNA was harvested from SW480-scramble and SW480-siMAD2B cells as in A and used for quantitative real-time PCR with primers for Slug and MAD2B. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase. The inset graph showed that MAD2B mRNA level was knocked down in SW480-siMAD2B cells. Data represent the mean ± S.D., n = 3. *, p < 0.05 compared with control. C, MAD2B silencing enhances the ability of TCF4 to bind the Slug promoter. SW480-scramble and SW480-siMAD2B cells were treated with formaldehyde and subjected to a ChIP assay with TCF4 antibodies. Precipitated genomic DNA was used for quantitative real-time PCR with primers for the human Slug promoter region. The real-time PCR target contained the consensus TBS, which is highly homologous with the TBS in chick and mouse Slug promoters (upper half of panel C). Conservative TBS was highlighted in bold. Normal mouse IgG was used as an antibody control. Data are presented as the percentage of input control. Experiments were performed in duplicate. *, p < 0.05 compared with control.

Model of functional Interplay between MAD2B and TCF4 in modulation of epithelial-mesenchymal transdifferentiation. TCF4-β-catenin complex binds the Slug promoter on TBS and transactivates Slug expression, which suppresses transcription of E-cadherin and induces EMT. Interaction of TCF4 with MAD2B inhibits the DNA binding ability of TCF4, which attenuates transactivation of Slug, thus releasing the suppression of E-cadherin and leading to mesenchymal-epithelial transdifferentiation (MET). β-CTN, β-catenin.