Effects of MMP8 mutations on enzymic activity, growth on soft agar and growth in vivo. (a) Collagen I zymography was used to assess activity of wild-type and altered MMP-8 proteins. Conditioned media from HEK 293T cells expressing wild-type MMP-8 or altered MMP-8 with the indicated amino-acid substitutions were immunoprecipitated and subjected to type I collagen substrate gel zymography. Each panel represents a separate experiment. (b) Anchorage-independent proliferation of Mel-STR cell clones expressing the indicated constructs was assessed by measuring colony growth in soft agar. Graph indicates number of colonies observed after two weeks of growth. (c) SK-Mel-2 cells were infected with either control shRNAs or two different shRNA constructs targeted against MMP8 (#1 and #5) and selected to form stable pooled clones. Left panel: conditioned media of the clones were immunoblotted with the indicated antibodies. Right panel: anchorage-independent proliferation of infected SK-Mel-2 cell clones expressing the indicated constructs was assessed as described in b. (d) NOD/SCID mice were subcutaneously injected with Mel-STR cell clones expressing the indicated constructs and examined on a bi-weekly basis (n=8; *P < 0.05 to **P < 0.005; Fisher’s exact test.) (e) Representative hematoxylin and eosin-stained images of histopathological pulmonary sections from mice injected with MMP-8 mutant clones. Top left: parenchymal metastasis which probably originated in the blood vessels (arrow heads); emphysematous areas are indentified with a black dot. Top right: pleural metastasis island associated with focal parenchymal collapse or atelectasia (arrow). Bottom left: parenchymal metastasis growing along a bronchial structure (arrows). Bottom right: perivascular metastasis (arrows) with surrounding emphysematous areas. All slides are at ×130. α-Tubulin, antibody to tubulin.