Figure 4. CtBP2 plays a proliferative role in breast cancer cells.

(A) Flow cytometry analysis of cell cycle progression in MDA-MB-231 cells. Cells that were synchronized at G1 progressed into the cell cycle 0, 2, 6, 12, and 24 h after serum stimulation. Finally, most of the cells entered S phase. (B) MDA-MB-231 cells were serum starved for 48 h (S48h). Upon serum stimulation, cell lysates were prepared and analyzed by Western blot using antibodies against CtBP2, p16^INK4A, PCNA and GAPDH. GAPDH was used as a control for loading and protein integrity. (C) The bar graph demonstrates the ratio of CtBP2, p16^INK4A and PCNA proteins to GAPDH at each time point, as determined by densitometry. The data are represented as the mean ± SEM (n = 3, *^{^#}P < 0.01, compared with control: S48 h). S: serum starvation; R: serum stimulation. (D) Light microscopy shows that pcDNA3.1-EGFP and pcDNA3.1-EGFP-CtBP2 are expressed in MDA-MB-231 cells. Proteins were analyzed by Western blot using an anti-EGFP antibody. (E) MDA-MB-231 and MCF-7 cells were transfected with pcDNA3.1-EGFP- CtBP2 or nothing (control). RT-PCR shows transcriptional levels of the p16^INK4A gene 48 h post-fection, and GAPDH was used as a loading control. The data are means ± SEM *^#P < 0.01, compared with the control group. (F) MDA-MB-231 and MCF-7 cells were transfected with the pcDNA3.1-EGFP-CtBP2, or control, as indicated. Protein expression of p16^INK4A and GAPDH was analyzed by Western blot. Data are presented as means±SEM *^#P < 0.01, compared with the control group. (G) MDA-MB-231 and MCF-7 cells were transfected with either pcDNA3.1-EGFP-CtBP2 or control. Cell growth of the transfected cells was assessed by the CCK-8 cell viability assay. The data are presented as the mean±standard error of three experiments. (H) Cell cycle analysis was performed by staining CtBP2 overexpressing MDA-MB-231 and MCF-7 cells with PI.