Fig 2: The expression of DEC1 in human breast tumor tissue samples and the effect of DEC1 on the cell proliferation. (a and b) Immunohistochemistry (IHC) analysis of DEC1 expression in adjacent normal breast tissues (n=18) and breast carcinoma (n=30). Each sample was incubated with antibody against DEC1. Positive staining and negative staining are indicated by brown and blue staining, respectively. (c and d) MCF-7 cells and T47D cells were transfected with Flag-DEC1 or control vectors (Ctrl). Cells were then cultured in selective medium (500 µg/ml G418) and subjected to colony formation and MTT assays (days, time after transfection). Each bar represents the mean±S.D. from five independent experiments. *P<0.05. (e) Western blot analysis of endogenous DEC1 expression. MCF-7 cells were transfected with DEC1 siRNAs (siRNA-1 and siRNA-2) for 24 h and probed with anti-DEC1 and anti-ß-actin. Each bar represents the mean±S.D. from three independent experiments. (**P<0.01). (f) Colony formation assay, MCF-7 cells were transfected with the shDEC1 vector, followed by 2-week selection with hygromycin B. Hygromycin-resistant clones are shown in the left panel. The right panel shows the corresponding quantitative analyses. Only representative data from three independent experiments are shown. Scale bar, 500 µm

Fig 3: DEC1 regulates the stability of cyclin E in MCF-7 via Fbw7-mediated cyclin E ubiquitin-proteasome pathway. (a and b) Western blot analysis of the effect of DEC1 on the half-life of cyclin E. MCF-7 cells were transfected with Myc-cyclin E and Flag-DEC1 or empty vector. The cells were then treated with CHX only (a) or both CHX and MG132 (b), and harvested at the indicated time periods. The cell extract was subjected to western blot with anti-Myc, anti-Flag and anti-ß-actin antibody. The histogram in the right panel (a and b) shows the quantitative analysis of cyclin E bands. (c) MCF-7 cells were transfected with HA-tagged cyclin E and Myc-Ub or empty vector, followed by treatment with MG132 for 8 h before harvest. The cell extract was immunoprecipitated with anti-HA antibody and then probed with anti-Myc antibody. (d) MCF-7 cells were transfected with plasmids expressing the indicated Flag-tagged Fbw7 isoforms together with Myc-tagged cyclin E or empty vector or DEC1, followed by western blot analysis with anti-Myc antibody. (e) MCF-7 cells were transfected with Myc-tagged cyclin E, Flag-Fbw7a and Flag-DEC1 or empty vector, followed by treatment with MG132. Clear cell extracts were probed with anti-Myc and anti-Flag antibodies. (f) MCF-7 cells were transfected with Myc-cyclin E only or together with Flag-DEC1 in the presence of sh-Fbw7 or sh-c. The histogram shows the quantitative analysis of cyclin E protein levels after normalization to ß-actin (bottom). Data are the means±S.D. (g) MCF-7 cells were transfected with Myc-tagged cyclin E, Flag-Fbw7a and GFP-DEC1 or empty vector, treated with MG132 for 8 h before harvest. The clear cell extracts were immunoprecipitated with anti-Myc antibody, and then probed with anti-Flag, anti-GFP and anti-Myc antibody as indicated. ß-Actin was used as a negative control. (h) Effect of DEC1 on the interaction between Fbw7a and cyclin E under serum starvation stress. MCF-7 cells were transfected as in (g), and cultured in serum-free medium, and then treated with 10 µM MG132 for 8 h before harvest. The cells were collected and subjected to immunoprecipitation assay as in (g). (i and j) Effect of DEC1 on the interaction between endogenous Fbw7a and cyclin E. MCF-7 cells were transfected with GFP-DEC1 or empty vector, and cultured with or without serum, and then treated with MG132 for 8 h before harvest. The clear cell extracts were immunoprecipitated with anti-IgG or anti-cyclin E antibody, probed with anti-Fbw7, anti-GFP and anti-cyclin E antibody as indicated. ß-Actin was used as a negative control. (k) Ubiquitination status of endogenous cyclin E in DEC1- and Fbw7-silenced cells. MCF-7 cells were transfected with either shDEC1 or shFbw7a, or both. Cell extract was immunoprecipitated with anti-cyclin E antibodies, followed by western blotting using anti-Ub or anti-cyclin E antibody. Total cell lysate was also analyzed by western blotting using anti-ß-actin antibody. To test the expression and immunoprecipitation of cyclin E, mouse monoclonal (SB62a) secondary antibody against rabbit IgG light chain (HRP, ab99697) was used at a 1 : 5000 dilution in (g–k)

Fig 4: DEC1 promotes the complex formation and activity of cyclin E/Cdk2 and inhibits cell cycle progression through the S phase. (a) Effect of DEC1 on the formation of cyclin E/Cdk2 complex. MCF-7 cells were transfected with HA-Cdk2 and Myc-cyclin E only or HA-Cdk2, Myc-cyclin E and Flag-DEC1, and the clear cell extract was immunoprecipitated with anti-Myc antibodies and then probed with anti-HA antibody. (b) Effect of DEC1 on the sequent formation of the cyclin A/Cdk2 complex as detected by CoIP. MCF-7 cells were transfected with empty vector only, HA-Cdk2 and Myc-cyclin A only or HA-Cdk2, Myc-cyclin A and Flag-DEC1, and the clear cell extract was immunoprecipitated with anti-Myc antibody and then probed with anti-HA antibody. (c) Mammalian two-hybrid assay analysis of the effect of DEC1 on the interaction between cyclin E and Cdk2. Cyclin E, Cdk2 and DEC1 were expressed from pBIND-cyclin E, pACT-Cdk2 and Flag-DEC1, respectively. Each bar represents the mean±S.D. from three independent experiments. **P<0.01 compared with cells transfected with pACT and pBIND. (d) Effect of DEC1 on the activity of Cdk2/cyclin E kinase. Columns, mean (n=3)±S.D.; *P<0.05 compared with controls. (e) Immunofluorescence assay showing the effect of DEC1 on the sublocation of cyclin E. MCF-7 cells were transfected with Myc-cyclin E only or Myc-cyclin E and Flag-DEC1, and were fixed and stained with rabbit anti-Myc antibody (red) and then counterstained with DAPI (blue) for nucleus detection. (f) Effect of DEC1 on the sublocation of Cdk2. MCF-7 cells were transfected with HA-Cdk2 only or HA-Cdk2 and Flag-DEC1 and stained with mouse anti-HA antibody (green), and then counterstained with DAPI (blue) for nucleus detection. (g and h) CoIP assay showing the interaction between DEC1 and Cdk2. Control cells or cells overexpressing HA-Cdk2 only, Flag-DEC1 only or Flag-DEC1 and HA-Cdk2 were subjected to immunoprecipitation with anti-HA antibody (g) and with anti-Flag antibody (h). (i) Mammalian two-hybrid assay analysis of the interaction between DEC1 and Cdk2. DEC1 and Cdk2 were expressed from pACT-DEC1 and pBIND-Cdk2, respectively. Columns, mean (n=3)±S.D.; **P<0.01 compared with controls

Fig 5: Interaction between DEC1 and cyclin E. (a) MCF-7 cells were transfected with Flag-tagged DEC1 and Myc-tagged cyclin E. After 24 h of transfection, the cell extract was subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis with anti-Myc antibody. (b) MCF-7 cells transfected as in (a) were subjected to serum starvation for 24 h. The cell extract was subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-Myc antibody. (c and d) CoIP experiment showing the interaction between endogenous DEC1 and cyclin E in serum-plus and serum-free conditions. MCF-7 cells were subjected to immunoprecipitation with anti-cyclin E antibody followed by western blot analysis with anti-DEC1 antibody. Immunoprecipitation carried out with anti-IgG antibody was used as control. Mouse monoclonal (SB62a) secondary antibody against rabbit IgG light chain (HRP) was used at a 1 : 5000 dilution. (e) Western blot analysis of GST pull-down assay showing the interaction between cyclin E and DEC1. The cell extract was incubated with glutathione-agarose beads coated with purified GST or GST-tagged cyclin E as indicated at the top. After extensive washing, bound proteins were eluted, resolved by SDS-PAGE, and probed with anti-DEC1 and anti-GST antibodies. (f and g) Interaction between DEC1 and cyclin E as detected by a mammalian two-hybrid system for cells without and with serum starvation. Cyclin E and DEC1 were expressed from pBIND-cyclin E and pACT-DEC1, respectively. The MCF-7 cells were transfected with pG5-luc and the empty vectors (pACT and pBIND) as indicated. Positive control cells were transfected with pBIND-ID and pACT-MyoD. Each bar represents the mean±S.D. from three independent experiments. **P<0.01 compared with cells transfected with pACT and pBIND. (h and i) Co-localization of DEC1 and cyclin E in MCF-7 cell nuclei. MCF-7 cells were transfected with Myc-cyclin E and Flag-DEC1. Flag-antibody complex and Myc-antibody complex were visualized with FITC and TRITC, respectively. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (j) CoIP experiments showing the dynamics of the interaction between DEC1 and cyclin E. MCF-7 cells were transfected with Flag-DEC1, and then subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-cyclin E antibody. HRP (ab99697) was used as secondary antibody