Fig. 3

MMP-8 expression alters α6β4 subcellular distribution and myoepithelial cell morphology. a (i) Confocal images of β6-1089 cells transfected with empty vector (EMPTY) wild-type matrix metalloproteinase-8 (MMP-8 WT) or inactive mutant MMP-8 (EA) and grown on Fibronectin for 24 hours then fixed and co-stained for α6β4 (red) and plectin (green). Insert: cartoon illustrating hemidesmosome (HD) components, indicating the integrin α6β4 associates with plectin to link the complex to the keratin cytoskeleton. (ii) Co-localisation of α6β4 and plectin was determined by quantifying the number of yellow pixels per field of view in each condition then expressed as a ratio as compared to EMPTY. β6-1089 transfected with WT demonstrated a significant increase in α6β4 and plectin co-localisation, while the β6-1089 transfected with EA exhibited no change as compared to EMPTY. b (i) Confocal images of β6-1089 cells transfected with empty vector (EMPTY) wild-type MMP-8 (WT) or inactive mutant MMP-8 (EA) and grown on Fibronectin for 24 hours then fixed and co-stained for α6β4 (green) and phallodin (red). (ii) Morphological features of the transfected β6-1089 were analysed and length of trailing fibres was noted to be altered. Analysis of the length of these fibres indicated that β6-1089 transfected with MMP-8 WT significantly reduced the fibre length as compared to β6-1089 transfected with empty vector or MMP-8 EA. (iii) Analysis of the number of these fibres per cell also demonstrated a significant reduction in β6-1089 transfected with MMP-8 WT as compared to β6-1089 transfected with empty vector or MMP-8 EA. (iv) The final morphological alteration analysed was the physical space the cells occupied, the relative area occupied by β6-1089 transfected with MMP-8 WT was significantly larger than that occupied by β6-1089 transfected with empty vector or MMP-8 EA. c (i) Confocal images of N-1089 cells transfected with siRNA targeting MMP-8 (siMMP-8) or Luciferase (siLuc) as a control grown on Fibronectin for 24 hours then fixed and co-stained for α6β4 (green) and phallodin (red). (ii) Trailing fibre length was significantly longer in N-1089 transfected with siMMP8 as compared to siLUC. (iii) There were significantly more trailing fibres per cell in the N-1089 + siMMP8 group as compared to N-1089 transfected with siLUC. ^* p = 0.05 ^** p = 0.01, ^*** p = 0.001 (Student’s t test). Error bars = SEM, n = 3 independent experiments