Figure 2: Filamin accumulation in the surrounding normal epithelial cells plays a crucial role in apical extrusion of transformed cells.
From: Filamin acts as a key regulator in epithelial defence against transformed cells
(a–c) Immunofluorescence images of filamin (red) in the mix culture of normal and ts-Src MDCK cells. ts-Src MDCK cells stained with CMFDA (green) were surrounded by normal MDCK cells. v-Src activation was induced at 35 °C (a,c) or not induced at 40.5 °C (b). The area in the white box is shown at higher magnification in the lower panel. Arrows indicate filamin accumulation. (d) Quantification of the filamin accumulation. Data are mean±s.e. from three independent experiments. *P=2.7 × 10−4 (Student’s t-test). (e) Immunofluorescence images of filamin (red) in the mix culture of Src-transformed cells stained with CMFDA (green) and non-labelled Src-transformed cells. (f) Immunofluorescence images of filamin (red) in the mix culture of normal and RasV12-MDCK cells. RasV12-MDCK cells stained with CMFDA (green) were surrounded by normal MDCK cells. GFP-RasV12 expression was not induced (left panels) or induced by tetracycline (right panels) The area in the white box is shown at higher magnification in the lower panel. Arrows indicate filamin accumulation. White dashed lines in xy panels denote the cross-sections represented in xz panels (c,f). (g) Effect of cytochalasin D on filamin accumulation in the surrounding normal cells at the interface with Src-transformed cells. ts-Src-MDCK cells stained with CMFDA (green) were mixed with normal cells, and were treated with dimethyl sulphoxide (DMSO) or cytochalasin D followed by immunofluorescence with anti-filamin antibody (red). Arrows or arrowheads indicate intact or disturbed filamin accumulation, respectively. Scale bars, 10 μm (a–c,e–g). (h) Establishment of MDCK cells stably expressing filamin A shRNA1 in a tetracycline-inducible manner. Knockdown was induced for 72 h with tetracycline treatment, followed by western blotting with the indicated antibodies. Note that there are three mammalian filamin homologues (filamin A, B and C), but the majority of filamin proteins in MDCK cells detected by the anti-filamin antibody is filamin A. (i,j) Effect of knockdown of filamin A in the surrounding cells on apical extrusion of Src- (i) or RasV12- (j) transformed cells. Data are mean±s.d. from three (i) or two (j) independent experiments. *P=1.2 × 10−4, **P=0.0031 (Student’s t-test).
