Fig 2: Upregulation of E2F1 promotes the expression of stathmin1 and TACC3 and facilitates cell cycle progression. A Total proteins were extracted from HT29, HCT8, DLD1, RKO, SW480, SW620, and HCT116 cells. The levels of E2F1, stathmin1, and TACC3 were determined by immunoblotting. B Correlations among E2F1, stathmin1, and TACC3 levels in the CRC cell lines shown in A. Pearson’s test was used to analyze the correlations. C Viability of the cells shown in A, as measured using MTS assays 0, 24, 48, and 72 h after cell seeding. D DLD1 cells were transfected with pCMV-E2F1 or pCMV-blank vectors (final vector concentrations, 2 μg/ml). HCT116 cells were transfected with E2F1-specific or control siRNA (final siRNA concentrations, 20 nM). Samples were harvested 48 h after transfection, and the levels of the indicated factors determined by immunoblotting. E Relative levels of expression of the proteins indicated in D. F DLD1 and HTC116 cells were transfected with vectors or siRNAs as described in D. Cell viability was measured using MTS assays 0, 24, 48, and 72 h after transfection

Fig 3: Phosphorylation of stathmin1 at Ser16 facilitates phosphorylation of TACC3 at Ser558. A Prediction of potential kinases targeting stathmin1 at Ser16 and TACC3 at Ser558 using the NetPhos 3.1 database. PKA was found to target both of these proteins at the indicated sites. B HCT116 cells were transfected with stathmin1-specific or control siRNAs (final concentrations, 20 nM). Cells were treated with 100 μM bucladesine 4 h before harvesting and harvested 48 h after transfection. The levels of indicated factors were determined by immunoblotting. C Relative levels of expression of the proteins indicated in B. D DLD1 cells were transfected with pCMV-stathmin-FLAG, pCMV-stathmin1-M-S16-Ala-FLAG, or pCMV-FLAG vector (final vector concentrations, 2 μg/ml) and harvested 48 h later transfection. The levels of the indicated factors were determined by immunoblotting. E Relative expression levels of the proteins indicated in D. F DLD1 cells were transfected with vectors as shown in D. Cell viability was measured using MTS assays 0, 24, 48, and 72 h after transfection. G DLD1 cells were transfected with vectors as shown in D. Cell cycle progression was analyzed by flow cytometry 48 h after transfection. H Percentage of cells in G in mitotic phase. I Representative images of tumors formed by DLD1 cells with indicated stable transfections. J The growth curves of the tumors formed by the indicated DLD1 cells (N = 6 mice per group). K Weight differences in tumors formed by the indicated DLD1 cells (N = 6 mice per group)

Fig 4: Stathmin1 positively regulates the phosphorylation of TACC3 at Ser558. A DLD1 cells were transfected with pCMV-stathmin1, pCMV-TACC3, or pCMV-blank vector (final vector concentrations, 2 μg/ml), and HCT116 cells were transfected with stathmin1-specific, TACC3-specific, or non-specific siRNAs (final siRNA concentrations, 20 nM). Cell viability 0, 24, 48, and 72 h after transfection was measured using MTS assays. B DLD1 and HTC116 cells were transfected with vectors or siRNAs as shown in A. Cell cycle progression was analyzed by flow cytometry 48 h after transfection. C Percentage of cells in B that were in mitotic phase. D DLD1 and HTC116 cells were transfected with vectors or siRNAs as shown in A. Samples were harvested 48 h later, and the levels of indicated factors were determined by immunoblotting. E Relative levels of expression of the proteins shown in D

Fig 5: E2F1 transactivates stathmin1 and TACC3 in CRC cells. A Schematic representations of the promoter regions of human STMN1 and TACC3. The predicted E2F1 binding sites are indicated. B Agarose gel electrophoresis of ChIP-PCR products in the input, IgG, and ChIP groups, showing that E2F1 interacted with the STMN1 promoter at P3, and with the TACC3 promoter at P2/P3. C Quantitative PCR analysis of the efficiency of interaction of E2F1 DNA at the promoter sites shown in B. D Luciferase activities of DLD1 cells transfected with pGP4.19 promoters carrying wild-type and mutant binding sites promoter, pRL-TK and vector/E2F1