Figure 1.
FBI-1 enhances transcriptional activity of ETS-1.
(A-D) Lovo cells were transfected with ETS-RE-Luc reporter. (A, C-D) Cells were stably transfected with FBI-1 expression vector or empty vector. (B) Cells were transfected with FBI-1 siRNA or control siRNA. (D) Cells were transfected with ETS-1 siRNA (D). Lovo cells were treated with 5 ng/ml HGF (A-D, F), or ARQ-197 (C). (E) Overexpression or knockdown of FBI-1 were examined by western blot. The luciferase values are the mean ± SE of three independent experiments with similar results. *P<0.05 versus with the empty vector or the FBI-1 vector (A-D); versus with the control siRNA or the FBI-1 siRNA (A-D); versus with or without HGF/ARQ-197.
Figure 2.
FBI-1 modulates ETS-1 downstream genes expression in Lovo cells which were stably transfected with plasmids.
(A-B) Western blotting with various antibodies showed the overexpression of FBI-1 or specific knockdown effect of FBI-1 siRNA on the endogenous FBI-1 protein level. Lovo cells were harvested for WB assays and detected by anti-FBI-1 antibody, anti-MMP1 antibody, anti-MMP9 antibody, anti-u-PA antibody, anti-c-Met antibody, anti-ETS-1 antibody, or anti-GAPDH antibody.
Figure 3.
FBI-1 would potentially interact with ETS-1 and P53 in vivo.
(A) Interaction of FLAG-FBI-1 with exogenous ETS-1 or p53 in vivo. Lovo cells were transfected with FLAG-tagged FBI-1 or FLAG empty vector. Then, cell lysates were immunoprecipitated (IP) by anti-FLAG beads, and the precipitates were then immunoblotted (IB) with anti-FLAG antibody, anti-ETS-1 antibody, or anti-p53 antibody. (B) Lovo cells were transfected with FLAG-ETS-1 vector or FLAG empty vector. The IP analysis was performed with anti-FLAG antibody, and the IB analysis was performed with anti-FLAG antibody, anti-FBI-1 antibody, or anti-P53 antibody.
Figure 4.
FBI-1 can enhance the recruitment of ETS-1 to the MMP1 promoter.
(A) Lovo cells stably transfected with FBI-1 or empty vector were prepared and subjected to ChIP by using IgG antibody (negative control) or antibodies for ETS-1, FBI-1 and p53. The Immunoprecipitated DNA fragment was quantified by real-time PCR assay. (B) Lovo cells, which were stably transfected with FBI-1 siRNA, or control siRNA, were harvested for the ChIP assays. The ChIP assays were performed with IgG antibody (negative control) or antibodies for ETS-1, FBI-1 and p53. *P<0.05 versus the empty vector or the FBI-1 vector (A); or versus the control siRNA or the FBI-1 siRNA (B). The cloned promoter region of MMP1 is showed above the figure.
Figure 5.
Effect of FBI-1 on ETS-1 cytoplasmic/nucleus localization.
(A–B) The cells were fractionated into cytoplasmic and nuclear fractions. The fractions were examined with anti-ETS-1 antibody, anti-P53 antibody and anti-FBI-1 antibody. The Lamin A/C and tubulin were used as the nuclear and cytoplasmic indicator, respectively.
Figure 6.
p53 participates in FBI-1 modulating ETS-1 activity.
(A–D) Lovo cells were transfected with FBI-1 expression vector or empty vector. (A–B) Lovo cells were co-transfected with MMP1-Luc vector, p53 siRNA, or control siRNA, p53 vector or empty vector as indicated. Following transfection, cells were harvested for the luciferase assay. The values are the mean ± SE of three independent experiments with similar results. (C–D) Lovo cells were co-transfected with p53 vector or empty vector, p53 siRNA or control siRNA. The ChIP assays were performed with IgG of anti-ETS-1 antibody. (E–F) The luciferase activities changing of EBS-luc, p21-luc and ARE-luc upon FBI-1 and p53 overexpression and knocking down. The values are the mean ± SE of three independent experiments with similar results. *P<0.05 versus the empty vector or the FBI-1 vector (A–D), *P<0.05 versus the empty vector or the p53 vector (A, C), *P<0.05 versus the p53 siRNA vector or the control siRNA vector (B, D).
Figure 7.
FBI-1 promotes colorectal carcinoma cells proliferation in vitro.
(A–B) LoVo, (C–D) HR8348, and (E–F) HT29 cells were stably transfected with the plasmids. Then, relative cell numbers were determined by the MTT assay. Relative cell numbers (A and B) shown are Mean± SD of triplicate measurements and have been repeated 3 times with similar O.D. value results. *P<0.05 versus the empty vector or the FBI-1 vector (A, C, E), *P<0.05 versus the FBI-1 siRNA vector or the control siRNA vector (B, D, F).
Figure 8.
FBI-1 promotes Lovo cells anchor-independent growth and invasion.
(A–B) Lovo cells were stably transfected with the FBI-1 expression vector or the empty vector, or the FBI-1 siRNA vector or the control siRNA vector. Colony number was shown in the photographs (B).*P<0.05 versus the empty vector or the FBI-1 vector (A–B), *P<0.05 versus the FBI-1 siRNA vector or the control siRNA vector (A–B).
Figure 9.
The proposed models for certain roles of FBI-1 function in Lovo cells.
ETS-1 would be activated in presence of HGF and be blocked by ARQ-197. FBI-1 may modulate ETS-1 activity through potential protein interaction, recruitment to endogenous MMP1 promoter, or cytoplasmic/nucleus translocation. The regulatory effect of FBI-1 on ETS-1 is also through regulating P53 activity.