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. 2013 Jul 25;109(4):1040–1050. doi: 10.1038/bjc.2013.420

Figure 7.

Figure 7

Pazopanib increases CB activation and enzymatic activity in BC cells. (A) Lysates from 5637 and J82 cell lines, treated with 20 μM pazopanib or vehicle for 24 h, were separated on 12% SDS–PAGE and probed with anti-CB or anti-GAPDH Abs, respectively. Densitometric analysis, expressed as the mean±s.d., was performed by comparing pazopanib- with vehicle-treated cells, *P<0.01. (B) CB activity was measured at 12 h after 20 μM pazopanib alone or in combination with 2.5 μM CA074Me or vehicle by using the fluorogenic peptide Z-Arg-Arg-AMC. The fluorescence of the hydrolysed 7-amino-4-methyl-coumarin was detected on a SpectraMax Gemini XPS microplate reader. The CB contribution to the proteolysis was evaluated by using the specific inhibitor CA074Me. Data shown are the mean±s.d. of three independent experiments. Densitometric analysis, expressed as the mean±s.d., was performed by comparing pazopanib- with vehicle-treated cells and pazopanib plus CA074Me- with pazopanib-treated cells, *P<0.01. (C) Cell growth of 5637 and J82 cells treated with 20 μM pazopanib alone or in combination with 2.5 μM CA074Me was evaluated by MTT assay at 48 h after treatment. Data shown are the mean±s.d. of three independent experiments. Statistical analysis was performed by comparing pazopanib plus CA074Me- with pazopanib-treated cells, *P<0.01.