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Fig. 2 | Breast Cancer Research

Fig. 2

From: Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function

Fig. 2

MMP-8 expression alters myoepithelial cell adhesion and migration to ECM proteins. a Upper panel: immunoprecipitation of matrix metalloproteinase-8 (MMP-8) from 20× concentrated conditioned media of β6-1089 transiently transfected with wild-type MMP-8 (WT), inactive point mutant of MMP-8 (EA) or empty vector (EMPTY). Conditioned media (CM) was incubated overnight with MMP-8 antibody (R& D Systems, mAb 908) or IgG (Sigma-Aldrich) and bound proteins were precipitated with Protein A beads (B) and unbound supernatant was also collected (UB). Samples were separated by 10% SDS-PAGE and probed with anti-V5 antibody directed to the V5 tag on the transfected MMP-8. Lower panel: Collagen-I zymography. CM was collected from the β6-1089 cells as described above and concentrated 20× using 3 K spin columns. Equal volumes were run by SDS-PAGE containing 3 mg of Collagen-I. The gel was incubated overnight in substrate solution and then stained with Coomassie. Only the lane containing CM from β6-1089 transfected with WT MMP-8 demonstrated a clear band indicating Collagen degradation at the same molecular weight as MMP-8. b (i) Adhesion to ECM proteins. β6-1089 cells transiently transfected with empty vector (EMPTY), wild-type MMP-8 (WT) or inactive mutant MMP-8 (EA) were plated on Fibronectin (Fib), Collagen-I (Col-I), Collagen-IV (Col-IV), Laminin-I (Lam-I), Tenascin-C (Ten-C), latency-associated peptide (LAP) or BSA (Control) for 1 hour. Then cells were treated with Calcein AM for 15 minutes before reading the fluorescence. The adhesion is calculated relative to the EMPTY. Expression of WT MMP-8 increased cell adhesion to Fib, Col-I, Lam-I and Ten-C, and decreased adhesion to LAP. The EA transfected cells did not show alterations in adhesion.(ii) β6-1089 cells treated in the same way as above were plated onto transwells coated on the underside with the same ECM proteins and cells were allowed to migrate for 8 hours before quantifying the number of cells that were on the top and bottom of the transwell. Migration was calculated relative to the EMPTY. Expression of WT MMP-8 decreased migration towards Fib, Col-I, Lam-I, Ten-C and LAP, while expression of the EA had no effect. c (i) N-1089 cells were transfected with siRNA targeting MMP-8 (siMMP-8) or Luciferase (siLuc) as a control. Ninety-six hours after transfection protein and RNA were collected and analysed by western blot and RT Q-PCR respectively. Both the western blot (upper panel) and RT Q-PCR (graph) indicate a reduction in MMP-8 at the protein and mRNA level in response to siMMP-8 treatment as compared to the siLUC control. (ii) N-1089 cells treated with siMMP-8 as described were plated onto the same selection of ECM proteins as previously described in (b). A significant reduction in adhesion to Fib, Col-I, Col-IV, Lam-I and Ten-C was observed in N-1089 transfected with siMMP-8 as compared to N-1089 transfected with siLUC. (iii) N-1089 cells treated with siMMP-8 were also plated on transwells coated with the same selection of ECM proteins as described in (b). The N-1089 transfected with siMMP-8 demonstrated a significant increase in migration towards Col-IV, Lam-I and Ten-C as compared to N-1089 + siLUC. ** p = 0.01, *** p = 0.001 (Student’s t test). Error bars = SEM, n = 3 independent experiments

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