Figure 1.
DDB2 is overexpressed in human ER-positive breast cancer cell lines.
(A) Total RNA was extracted from cells, then subjected to semiquantitative RT-PCR analysis. The relative levels of DDB1 and DDB2 mRNAs were normalized to those of β-actin mRNA. (B) DDB1 and DDB2 proteins were analysed in the total protein (50 µg) extracted from cells by Western blotting, using polyclonal anti-DDB1 and anti-DDB2 antibodies. Coomassie blue membrane staining was used as the protein loading control. Results are representative of three independent experiments. Relative band intensities for RT-PCR and Western blot analysis were quantified by densitometry. Data from RT-PCR analysis are expressed as the ratio for DDB1 or DDB2 mRNA levels/ β-actin mRNA levels. Data from Western blot analysis are expressed as relative densitometric units. Statistically significant differences from the HMEC values for DDB1 and DDB2 are indicated as ** and *P<0.05, respectively.
Figure 2.
DDB2 is expressed in human breast tumors from patients.
Total RNA was extracted from eight ER-positive and eight ER-negative breast cancer samples, then subjected to semiquantitative RT-PCR analysis. The relative levels of DDB1 and DDB2 mRNAs were normalized to those of β-actin mRNA. Statistically significant differences between ER-positive and ER-negative samples are indicated as P<0.05. The mean values are indicated by a bar in the graph for each group of tumors and DDB1 or DDB2 mRNA levels.
Figure 3.
DDB2 knockdown affects the growth and colony formation of MCF-7 cells.
(A) Generation of MCF-7 cell clones stably expressing DDB2 siRNA. Total RNA was extracted from parental MCF-7 cells (Wt) and from cells stably transfected with either the DDB2-siRNA vector (clones cl.2 and cl.3) or the scrambled siRNA vector (control siRNA), then subjected to RT-PCR analysis. The relative levels of DDB1, DDB2, DHFR, cyclin E and PCNA mRNAs were normalized to those of β-actin mRNA. DDB1 and DDB2 protein levels were analysed in the total protein (50 µg) extracted from MCF-7 cell clones stably expressing DDB2 siRNA and from control cells, by Western blotting using polyclonal anti-DDB1 and anti-DDB2 antibodies. Coomassie blue membrane staining was used as the protein loading control for Western blot analysis. (B) Parent cells (Wt), control siRNA-transfected MCF-7 cells and the two DDB2 siRNA-transfected cell clones (DDB2 siRNA cl.2 and cl.3) were plated in 24-well dishes (1×104 per well) in complete medium and cell numbers were counted for 4 days. Means are shown for three experiments. Cell population doubling time was calculated from the cell growth curve during the exponential growth phase. (C) Wt cells, control siRNA and DDB2 siRNA transfected cells were seeded (500 cells) in 100-mm dishes and grown for 12 days. Colonies with more than 50 cells were counted and data from three independent experiments were expressed as the % of colony formation = (colonies formed/cells seeded)×100%. Statistically significant differences from the parental (Wt) cell value are indicated as * P<0.05.
Figure 4.
DDB2 overexpression increases the growth and colony formation of MDA-MB231 cells.
(A) MDA-MB231 cells were stably transfected with the pcDNA 3(+) expression vector containing either DDB2 cDNA (MDA DDB2) or no insert (MDA Neo). The DDB2 protein level was assessed by Western blot analysis, using equal amounts of protein (50 µg) extracted from parent cells (MDA Wt), empty vector-transfected cells (MDA Neo) and DDB2-overexpressing cells (MDA DDB2), and using DDB2 polyclonal antibody. Coomassie blue membrane staining was used as the protein loading control. (B) Parent cells (MDA Wt), empty vector-transfected cells (MDA Neo) and DDB2-transfected cell clone (MDA DDB2) were plated in 24-well dishes (1×104 per well) and cultured in complete medium. Cell numbers were counted on days 1, 3, 5, 7 and 9. Means are shown for three experiments. Cell population doubling times were calculated from the cell growth curves during the exponential growth phase. (C) MDA Wt, MDA Neo and MDA DDB2 cells were seeded (500 cells) in 100-mm dishes and grown for 12 days. Colonies with more than 50 cells were counted and data from three independent experiments were expressed as the % of colony formation = (colonies formed/cells seeded)×100%. Statistically significant differences from the MDA Wt cell value are indicated as * P<0.05.
Figure 5.
DDB2 knockdown affects cell cycle distribution of MCF-7 cells.
Parent cells (Wt), control siRNA-transfected MCF-7 cells and the two DDB2 siRNA-transfected cell clones (DDB2 siRNA cl.2 and cl.3) were synchronized by serum starvation for 48h, and induced to re-enter the cell cycle by the addition of serum for 0, 3, 12 or 18h. MCF-7 cells were harvested for propidium iodide staining and analysed by FACS to determine the cell cycle fraction. FACS plots and data are representative of at least three separate experiments.
Figure 6.
DDB2 knockdown decreases the ability of MCF-7 cells to re-enter the S phase of the cell cycle.
Parent cells (Wt), control siRNA-transfected MCF-7 cells and the two DDB2 siRNA-transfected cell clones (DDB2 siRNA cl.2 and cl.3) were synchronized by serum starvation for 48h, and induced to re-enter cell cycle by the addition of serum for 0, 3, 12 or 18h. At the end of each of these periods after serum addition, MCF-7 cells were exposed to 5 BrdU for 20 min and were harvested for propidium iodide staining before being analysed by FACS for determination of the G1/S subpopulation and the S-phase fraction. FACS plots and data are representative of at least three separate experiments.
Figure 7.
Analysis of PCNA expression in DDB2-deficient MCF-7 cells.
Parent cells (Wt), control siRNA-transfected MCF-7 cells and the two DDB2 siRNA-transfected cell clones (DDB2 siRNA cl.2 and cl.3) were synchronized by serum starvation for 48h, and induced to re-enter the cell cycle by the addition of serum for 3, 12 or 18h. PCNA protein levels were analysed in the total protein (50 µg) extracted from different cells by Western blotting, using specific polyclonal antibodies. Membranes were then probed with specific polyclonal antibodies against tubulin or stained with Coomassie blue as the protein loading control for Western blot analysis.