Figure 7

Increased levels of TIMP1, CD63 and β1-integrins in melanoma cells compared to primary melanocytes and association between Timp1 and β1- integrins only in human metastatic melanoma cells. mRNA levels of TIMP1 and CD63 were measured by real time PCR (A and B, respectively). C. β1-integrin expression was analyzed in primary human melanocytes MP#2 and human metastatic melanoma cell lines (Mel2, Mel3, Mel4, Mel14, Mel25 and Mel28) by Western blotting using a specific polyclonal antibody. The same membrane was reprobed with anti-PCNA antibody as an internal control. D. Membrane-enriched protein extracts from MP#2, Mel2, Mel3 and Mel14 cell lines were immunoprecipitated with anti-TIMP1 and analyzed by Western blotting with anti-β1-integrin. E and F. Primary human MP#2 melanocytes and human metastatic melanomas Mel2, Mel3, Mel4, Mel11, Mel25, Mel28 and Mel33 (2x10²) were incubated at 37°C on 60 mm plates and colony formation capacity was determined after 5 days. Spearman’s correlation (r) was used to correlate TIMP1 and clonogenic capacity (G), CD63 expression and clonogenic capacity (H), TIMP1 and CD63 expression (I), and combined TIMP1 and CD63 expression and clonogenic capacity (J). All analyses were performed using SPSS (v 18, Chicago, IL, USA). The significance level was set at 0.05.