Fig. 6

MMP-8 expression in myoepithelial cells alters MMP-9 expression/function and TGF-β signalling. 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 rhodamine-tagged gelatin (red) for 24 hours then fixed and stained with FITC-conjugated phallodin (green). (ii) Gelatinase activity was quantified by measuring the total size of the black spots (degraded gelatin) per field of view relative to the β6-1089 transfected with EMPTY. There was a significant reduction in gelatin degradation in β6-1089 transfected with WT and EA compared to β6-1089 transfected with EMPTY, albeit the reduction in β6-1089 transfected with EA was not as marked. (iii) The number of cells per field of view was determined and no significant difference was noted between the groups. b (i) Confocal images of N-1089 cells transfected with siRNA targeting MMP-8 (siMMP-8) or Luciferase (siLuc) and grown on rhodamine-tagged gelatin (red) for 24 hours then fixed and stained with FITC-conjugated phallodin (green). (ii) Gelatin degradation was significantly increased in N-1089 transfected with siMMP-8 as compared to siLUC. (iii) Quantification of the number of cells per field of view indicated no difference between the two groups. c Confocal images of N-1089 cells transfected with siRNA targeting MMP-8 (siMMP-8) and MMP-8 and MMP-9 (siMMP-8/9) or Luciferase (siLuc) and grown on rhodamine-tagged gelatin (red) for 24 hours with or without an MMP-9 inhibitor at increasing concentration then fixed and stained with FITC-conjugated phallodin (green). The graph indicates relative gelatin degradation increases in N-1089 transfected with siMMP-8, this increase is significantly reduced when both MMP-8 and MMP-9 are knocked down (siMMP-8/9). The increase in gelatin degradation can also be significantly reduced, in a dose-dependent manner, with an MMP-9 inhibitor. d (i) Western blots for SMAD-2 on protein extracted from β6-1089 cells transfected with empty vector (EMPTY), wild-type MMP-8 (WT) or inactive mutant MMP-8 (EA) after treatment with TGF-β (5 ng/ml) for 5, 10 and 15 minutes. β6-1089 transfected with empty vector exhibit higher levels of Phospho (p)SMAD-2 at all time points than the β6-1089 transfected with WT, while the β6-1089 transfected with EA appear to show moderate expression. Total (t)SMAD-2 levels are similar in all lanes as is the actin loading control. (ii) Western blots for SMAD-2 on protein extracted from of N-1089 cells transfected with siRNA targeting MMP-8 (siMMP-8) or Luciferase (siLuc) after treatment with TGF-β (5 ng/ml) for 5, 10 and 15 minutes. N-1089 transfected with siMMP-8 exhibit higher expression of pSMAD-2 at 10 and 15 minutes than the N-1089 transfected with siLUC. The tSMAD2 and actin expression levels appear to be consistent. ^* p = 0.05 ^** p = 0.01, ^*** p = 0.001 (Student’s t test). Error bars = SEM, n = 3 independent experiments