Statistical analysis All tests were conducted in biological triplicate. The selleckbio re sults were organized into a database using the statistical program GraphPad Prism 5. The level of significance utilized was p 0. 05. The statistical tests performed were Students T test for Inhibitors,Modulators,Libraries unpaired samples, One way ANOVA followed by Tukeys multiple comparison tests and Spearmans Rank Correlation. Results Increased levels of membrane bound and secreted Timp1 in cells representing melanoma progression Previous data from our laboratory have shown signifi cant and progressive increase in the expression of timp1 in cells representing different phases of melanoma pro gression. This increase was accompanied by anoikis re sistance and more aggressive phenotype in vivo.
In agreement with our previous data, a progressive increase of Timp1 mRNA expression was found in 4C11 and 4C11 melanoma cell lines, as described for other melanoma cell lines from our model. To analyze the Timp1 protein expression and especially its association with cell surface, membrane enriched ex tracts of melan a, 4C, 4C11 and Inhibitors,Modulators,Libraries 4C11 cell lines were separated by SDSPAGE. Timp1 protein expression was observed in all cell linesderived from anchorage impediment of melan a melanocytes. Soluble Timp1 was found in the conditioned media from pre malignant 4C, and 4C11 and 4C11 melanoma cell lines, but not in that from non tumorigenic melan a melanocytes. Although the most known function of Timp1 is to inhibit matrix metalloproteinases, no correlation between Timp1 ex pression and MMPs activity was observed in our model, since Timp1 expressing melanoma cells presented high MMP activity.
Figure 1E Inhibitors,Modulators,Libraries shows that cell lines representing melanoma progression and expressing high levels of Timp1 present increased capability to form colonies compared to melan a melanocytes. Inhibitors,Modulators,Libraries Timp1 associates with B1 integrin and CD63 along melan a melanocyte malignant transformation Based on its importance and well established role, B1 integrin expression profile was first investigated in our model by Western Blot. A differential migration shift was observed in the bands corresponding to B1 integrin along melanoma Inhibitors,Modulators,Libraries genesis. Inter estingly, metastatic 4C11 melanoma cell line showed higher proportion between high and low molecular mass bands compared to melan a melanocytes, pre malignant 4C cells and non metastatic 4C11 melanoma cell line.
The high thenthereby molecular mass band corresponds to the mature and highly glycosylated form of B1 integrin and its increase in relation to the immature form is in agreement with the literature, since B1 integrins may present aberrant glycosylation in tumorigenic cells. CD63 expression, mainly on the cell surface, was eval uated in melan a, 4C, 4C11 and 4C11 cell lines, by flow cytometry using a specific polyclonal antibody against CD63.