Figure 1.
Expression of 53BP1 correlates with 5-Fu sensitivity in breast cancer cell lines.
(A) Expression mRNA levels and protein levels of 53BP1 in breast cancer cell lines. GAPDH or actin was used as the endogenous control. (B) The sensitivity of breast cell lines to 5-Fu were tested with MTT assay. The data were expressed as mean ± SD. (C) The localization of 53BP1 in breast cancer before and after the treatment of 5-Fu (5 uM). The immunofluorescent images of cells were stained with 53BP1 antibody. The nuclear staining with Nuclei was counterstained with DAPI. Red, 53BP1; Blue, DAPI. (D) The localization of H2AX in breast cancer before and after the treatment of 5-Fu. The immunofluorescent images of cells were stained with H2AX antibody. The nuclear staining with Nuclei was counterstained with DAPI. Red, H2AX; Blue, DAPI.
Figure 2.
53BP1 sensitizes breast cancer cells to 5-Fu in vitro.
(A) The transfection efficiency was measured by western blot analysis (left) and qRT-PCR (right). Results are shown for one of the three independent experiments performed. **p<0.01, ***p<0.001. (B) MDA-MB-231 control and 53BP1 overexpressing cells were treated with 5-Fu for 24 h (left) or 48 h (right). The effect of 53BP1 on 5-Fu sensitivity was measured by MTT assay. (C) MCF-7 control and 53BP1 knockdown cells were treated with 5-Fu for 24 h (left) or 48 h (right). The effect of 53BP1 on 5-Fu sensitivity was measured by MTT assay. The relative cell viability percentage is expressed as (absorbance of treated wells/absorbance of untreated wells).
Table 1.
Effect of 5-Fu on cell viability assessed by the MTT method (uM).
Figure 3.
53BP1 induced G2/M cell cycle arrest in breast cancer cells by flow cytometry analysis.
(A) The effect of 53BP1 overexpression on cell cycle in MDA-MB-231 cells. (B) The effect of 53BP1 knockdown on cell cycle in MCF-7 cells.
Figure 4.
53BP1 sensitizes 5-Fu-induced apoptosis in breast cancer cells.
The apoptosis related markers Akt, p-Akt (Ser 473), Bcl-2, Bax and P21 were detected by western blot in 53BP1 transfected MDA-MB-231 and MCF-7 cells. Results are shown for one of the three independent experiments performed.
Figure 5.
53BP1 sensitizes breast cancer to 5-Fu in vivo.
(A) Growth curves of mammary tumors for control and 53BP1 overexpressing MDA-MB-231 cells with 5-Fu or vehicle. (B) Growth curves of tumors for control and 53BP1 knockdown MCF-7 cells with 5-Fu or vehicle. Tumor diameter was measured with calipers and the tumor volume was calculated by the formula: (width)2 × length/2. Each group consisted of seven mice. Error bars represent ± SD. (C) Representative tumors isolated from mice after implantation with control or 53BP1 over expressing MDA-MB-231 cells and treated with 5-Fu or vehicle as described in panel A. (D) Representative tumors isolated from mice after implantation with control or 53BP1 knockdown MCF-7 cells and treated with 5-Fu or vehicle as described in panel B.
Figure 6.
53BP1 sensitizes 5-Fu-induced apoptosis in vivo.
(A) Representative TUNEL positive staining (red fluorescence) for control and 53BP1 overexpressing MDA-MB-231 xenografts. (B) The summary graph for (A). (C) Representative TUNEL positive staining for control and 53BP1 knockdown MCF-7 xenografts. (D) The summary graph for (B). *P<0.05; **P<0.01, ***P<0.001.
Figure 7.
53BP1 regulates the levels of TS and DPYD.
(A) The mRNA levels of TS in 53BP1 transfected MDA-MB-231 and MCF-7 cells. (B) The mRNA levels of DPYD in 53BP1 transfected cells. (C) The protein levels of TS and DPYD in 53BP1 transfected cells. (D) The transfection efficiency of siRNA TS was confirmed by western blot analysis. (E) The transfection efficiency of siRNA DPYD was confirmed by western blot analysis. Results are shown for one of the three independent experiments performed. **p<0.01, ***p<0.001.
Figure 8.
53BP1 sensitizes breast cancer cells to 5-Fu through TS and DPYD.
(A) The sensitivity of MCF-7 control and 53BP1 knockdown MCF-7 cells to 5-Fu after knockdown of TS and DPYD at the indicated time point. MCF-7 control cells, left; 53BP1 knockdown MCF-7 cells, right. (B) When the MCF-7 control and 53BP1 knockdown MCF-7 cells were treated for 48 h, the relative survival of each breast cell lines was tested with MTT assay.
Table 2.
Effect of 5-Fu on cell viability assessed by the MTT method (uM).