Figure 1.
FOSL2 is required for TGF-β1-induced migration.
(A) A549 cells were incubated with TGF-β1 (2 ng/ml) for the indicated times, and the cells were collected for western blot analysis. GAPDH was used as a loading control. (B) FOSL2 levels were examined by western blotting in FOSL2-siRNA and sicontrol A549 cells. GAPDH was also determined as a loading control. (C) Cell migration was measured using Transwell assays in sicontrol and siFOSL2 A549 cells with or without TGF-β1 (2 ng/ml) treatment. The cells migrating to the lower surface of the Transwell filters were photographed (top) and counted (bottom). (D) siControl and siFOSL2 A549 cells were incubated with or without 2 ng/ml of TGF-β1 for 72 h. Phase-contrast microscopy shows the cell morphological changes.
Figure 2.
(A) FOSL2 interacts with Smad3 in vivo. FLAG-FOSL2 and Myc-Smad3 were co-transfected into HEK293T cells. Cell lysates were harvested, and IP was performed with an anti-Myc antibody. FOSL2 and Smad3 were detected from the immunoprecipitates by western blotting with the indicated antibodies. (B) The association between endogenous FOSL2 and Smad3 in A549 cells with TGF-β1 (2 ng/ml) treatment. Immunoprecipitation was performed using an anti-IgG antibody or anti-Smad3 antibody. (C) Subcellular colocalisation of FOSL2 and Smad3 in A549 cells in the presence TGF-β1 (2 ng/ml). The nuclei were stained with DAPI. (D) Equivalent amounts of GST-Smad3 or GST alone were incubated with cell lysates from HEK 293T cells overexpressing Flag-FOSL2; 10% of the input was run on the gel as a control. The GST-Smad3 was detected by staining gels with Coomassie Blue.
Figure 3.
FOSL2 modulates TGF-β1-induced signalling responses.
(A) HEK 293T cells were transfected with the reporter p3TP-Lux plasmid harbouring Smad3 in the absence and presence of FOSL2, as indicated. The cells were harvested at 36 h after transfection and assayed for luciferase activity. The data are the means ± s.d. (B) A549 cells were transfected with siFOSL2 and control scrambled siRNA. After 24 h, the cells were transfected with p3TP-Lux and Smad3, as indicted. Luciferase activity was assayed after a further 24 h. The data are means ± s.d. (C) A549 cells transfected with siFOSL2 and control scrambled siRNA were stimulated with 2 ng/ml of TGF-β1 for 4 h. Total mRNAs were analysed by qRT-PCR using primers specific for p21 and PAI-1. The data are means ± s.d. (D) A549 cells transfected with siFOSL2 and control scrambled siRNA were stimulated with 2 ng/ml of TGF-β1 for 4 h and then harvested to produce cell lysates. The expression levels of the indicated proteins were examined by western blotting with the appropriate antibodies, as indicated.
Figure 4.
FOSL2 promotes P300 binding to Smad3 and the acetylation of Smad3 by P300.
(A) HEK293T cells were cotransfected with expression plasmids for HA-p300 and FLAG-FOSL2, together with Myc-Smad3, as indicated, with or without with TGF-β1 (2 ng/ml) treatment. Smad3-bound p300 was immunoprecipitated with an anti-Myc antibody and detected by western blotting with an anti-HA antibody. (B) A549 cells were transfected with siFOSL2. After 24 h, the cells were treated, as indicated, with TGF-β1 (2 ng/ml) treatment. (C) HEK293T cells were transiently transfected with the indicated plasmids. The cells were incubated in the presence of TSA (5 µM) for 20 h. Cell lysates were immunoprecipitated (IP) with an anti-Myc antibody, and acetylated Smad3 (Ac-Smad3) was detected by western blotting with an anti-acetylated lysine antibody. (D) A549 cells were transiently transfected with the indicated plasmids. The experiments were performed as described in Fig. 4C.
Figure 5.
Correlation of FOSL2 and activated Smad3 expression in NSCLC tumours.
(A) Immunohistochemical analysis of FOSL2 and p-Smad3 in 57 NSCLC samples. (B) FOSL2 expression is positively correlated with p-Smad3 expression in NSCLC samples. Semiquantitative scoring was performed (Pearson correlation test; r = 0.858, p<0.001). Note that some of the dots on the graphs represent more than one specimen (some scores overlapped). (C) Kaplan-Meier survival curves for NSCLC patients with FOSL2 higher or lower expression. The difference in postoperative survival between NSCLC patients with FOSL2 higher expression and with FOSL2 lower expression was greatly significant (P = 0.0059 by log-rank test).