FIG. 8.

Increased Ln-5 processing in Src-A cells. (A) MT1-MMP (green) and nuclei (blue) were visualized by IF microscopy of nonpermeabilized Src-L or Src-A cells at 3 h after cells were seeded onto BM in starvation medium. (B) The MCF-10A, Src-L, and Src-A cells were seeded, and Ln-5 was visualized as described in the legend to panel A (Ln-5). Grayscale Ln-5 fluorescence was inverted and overlaid onto bright-field images (overlay). Areas with decreased Ln-5 are in red. (C) Schema of human Ln-5 γ2 chain with the putative MT1-MMP cleavage site within laminin-type EGF-like subdomains of domain III (according to rat Ln-5 γ2 chain [46]). (D) Wbs of the Ln-5 γ2 p155 and p105 chains in total MCF-10A, Src-L, and Src-A cell extracts using anti-Ln-5 antibody. Expression controls for Src and actin are shown. Pixel densities of the p155 and p105 bands from three independent experiments were quantified, and the ratio of p105/p155 was determined (error bars are standard errors of the means). (E) Gelatin zymography using supernatants of the MCF-10A, Src-L, and Src-A cells grown for 18 h in complete or starvation medium. (F) Gelatin zymography and Wb using membrane extracts of the MCF-10A, Src-L, and Src-A cells grown for 18 h in complete medium. SDS-PAGE was performed under nondenaturing conditions. (G) Ln-5 (red), MT1-MMP (green), and nuclei (blue) were visualized in 11-day acinar cultures by IF confocal microscopy. Bars correspond to 20 μm, unless otherwise stated.