Figure 4

EMT and stem cell properties of HMLER cells expressing FOXC2(S367) mutants. (a) Recombinant GST-FOXC2 fusion proteins: N-terminally truncated FOXC2 (amino acids 245–501), C-terminally truncated FOXC2 (amino acids 1–244), or N-terminally truncated FOXC2 (amino acids 245–501) with alanine substitution at serine 367 (S367A), were purified from E. coli using glutathione-sepharose-4B beads. The respective eluates were subjected to in vitro kinase assays with recombinant active p38. The reaction mixtures were resolved by SDS–PAGE, and the phosphorylated proteins were visualized by autoradiography. The electrophoretic mobility of phosphorylated GST-FOXC2 is indicated with an arrowhead. The GST control and the C-terminally truncated FOXC2 (amino acids 1–244), devoid of the putative p38 phosphorylation site, did not show any phosphorylation in this assay. The bottom panel depicts Coomassie blue staining of the protein input. (b) HMLER cells were transduced with empty vector, FOXC2, FOXC2(S367E) or FOXC2(S367A), and their morphology was imaged through phase-contrast microscopy. Scale bar, 100 μm. (c) Cell lysates from HMLER cells, transduced with the indicated constructs, were analyzed by immunoblotting for FOXC2 (anti-HA), E-cadherin, fibronectin and vimentin. β-Actin was used as a loading control. (d) Sphere formation by HMLER cells, transduced with the indicated constructs, is represented as the mean number of spheres formed/1000 seeded cells±s.e.m. (e) The indicated HMLER cells were analyzed by fluorescence-activated cell sorting for the presence of CD44 and CD24 on the cell surface. The circles denote the position of the vector-transduced control population in the cytograms. (f) Sphere formation by the indicated HMLER cells, treated with vehicle or SB203580, is represented as the mean number of spheres formed/1000 seeded cells±s.e.m. (g) The relative wound closure by the indicated HMLER cells, treated with vehicle or SB203580, was measured by image analysis and represented in a graphical format. Data are presented as mean±s.e.m. P-values were calculated using Student’s unpaired two-tailed t-test. *P<0.05; **P<0.01; ***P<0.001 compared with the control. n.s., not significant.