Figure 5.

Cancer-associated BSG mutants are differentially shed by ADAM12. (A) Illustration of cancer-associated mutations (marked in grey) in the extracellular membrane proximal region (aa. 190–208) of BSG, identified in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (BSG, ENST00000333511; August 2017). (B) Fold shedding in 293-VnR cells transfected with A12 or the catalytically inactive A12-E/A mutant together with wildtype (BSG-AP), or various BSG-AP point mutations identified in (A). (C) Western blot of AP in total cell lysates (TCL) from cells in (B). Actin was used as loading control. (D) In situ gelatinase assay, using HT1080 cells grown on Oregon green-labeled gelatin and treated with conditioned media from untransfected 293-VnR cells (control) or 293-VnR cells transfected with ADAM12 together with a full-length BSG expression construct harboring the A207T mutation. Dark areas (without green fluorescence) represent gelatin degradation, scale bar = 10 µm. (E) Gelatin degradation was measured in µm² for 27 images, using MetaMorph software and the percentage degradation was calculated. For both graphs, values represent means ± SEM from three independent experiments. * p < 0.05, Student’s t-test or ANOVA.