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Figure 4 | Molecular Cancer

Figure 4

From: c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

Figure 4

c-Myb upregulates cathepsin D and MMP9 and downregulates MMP1 in MDA-MB-231MYBup cells. (A) The relative amounts of MMP2, 3, 7, 10, 11, and 13 mRNA in the MYBup 5 and myb-less vector-transfected cells were determined by qRT-PCR. The relative amounts of MMP1, 9 and cathepsin D mRNAs were also determined in the MYBup 2 clone. Marks * and ** indicate significant (p < 0.05 and p < 0.01, respectively) differences in the relative amounts of cathepsin D, MMP9, and MMP1 mRNA in the myb-less vector-transfected and MYBup cells as determined by the t-test. The columns show the average values of relative normalized expression of the indicated genes from at least three independent experiments. (B) The control (wt, vector) and the MYBup cell protein extracts were analyzed by immunoblotting with anti-MMP1, anti-MMP9, and anti-cathepsin D antibodies. The secretion of cathepsin D, MMP9, and MMP1 was determined in the cell-conditioned medium. Equal amounts of total proteins were loaded. (C) To determine the activity of cathepsin D, protein extracts were prepared from harvested cells as described in the Material and Methods. The extent of hydrolysis of the fluorimetric substrate was measured in real time using a Synergy HT microplate reader. To demonstrate the assay specificity, pepstatin A was added to inhibit cathepsin D activity (data shown for MYBup 5 cells). (D) Transactivation assays were performed using six variants of luciferase reporters: two for cathepsin D (either containing the 2.85-kb or 0.82-kb promoter sequence), one for MMP9 (1.38 kb), and three for MMP1 (0.624, 1.606, and 4.3 kb). The luciferase activity of each sample was expressed in relative light units and normalized for transfection efficiency according to the β-galactosidase activity. The columns show the average values of relative luciferase activity from three independent measurements. Marks * and ** indicate significant (p < 0.05 and p < 0.01, respectively) differences in the luciferase activities in control cells and MYBup cells as determined by the t-test.

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