Figure 5.

p38-mediated phosphorylation of FOXC2 at S367 regulates metastasis. (a) 4T1 cells, transduced with either empty vector (pMIG) or FOXC2(S367E), were treated with vehicle or SB203580. Cell lysates were analyzed by immunoblotting for FOXC2 (using the rat anti-mouse FOXC2 antibody that also crossreacts with human FOXC2), with β-actin as a loading control. (b) 4T1 cells, transduced with empty vector or FOXC2(S367E), were subjected to a sphere-formation assay in the presence of vehicle or SB203580. Data are presented as the mean number of spheres formed/1000 seeded cells±s.e.m. (c) 4T1-vector or 4T1-FOXC2(S367E) luciferase-labeled cells were orthotopically injected into mice. Mice were treated daily with vehicle or SB203580, and primary tumor growth was monitored weekly by bioluminescence. Data are presented as the total photon flux plotted over time and reported as the mean±s.e.m. (d) Representative bioluminescent images of lungs harvested from mice in panel (c) at 4 weeks post-implantation. (e) The bioluminescent signal from the lungs harvested from mice in panel (c) was quantified to determine the incidence of metastasis. Data are reported as mean±s.e.m. P-values were calculated using Student's unpaired two-tailed t-test.*P<0.05; ***P<0.001 compared with the control. n.s., not significant.