Primers selleckbio were designed for hK1: Forward 5��-GGGTCGCCACAACTTGTTTG-3�� and Reverse 5��-GCTGTAGTCCTCGTCTGCTT-3�� B1R: Forward 5��-CTTCCCTCAAAATGCTACGGC-3�� and Reverse 5��-TCTGCCACGTTCAGTTGCC-3�� B2R: Forward 5��-GTCTGTTCGTGAGGACTCCG-3�� and Reverse 5��-CTGGGCAAAGGTCCCGTTAAG-3�� ACE: Forward 5��-AACGAAACCCACTTTGATGC-3�� and Reverse 5��-TCAGCCTCATCAGTCACCAG-3�� HMK: Forward 5��-TGGGGCCATGAAAAACAAAG-3�� and Reverse 5��-CTTGGCTAGGGAAGGGATGG-3�� LMK: Forward 5��-CCAGCATCTGAGAGGGAGGT-3�� and Reverse 5��-GCAGAATGGGTAGGGCTGAA-3��. BrdU cell proliferation assay Gastrointestinal stromal tumour cells were seeded in 96-well plates (5000 cells per well) and incubated with BrdU for 24h in normal medium or in the presence of 0.5, 0.05 or 0.005�� of specific hK1 inhibitors VA999154 or VA999024, kindly provided by Vantia Ltd.
(Chilworth, UK) BrdU incorporation was determined following the manufacturer’s protocol (Roche, Melwin, UK). GIST invasion assay The invasion capacity of GIST882 and GIST48 cells was measured by seeding 5 �� 104 cells on top of an 8��m filter coated with Matrigel (BD, 1:100). Medium containing 5% FBS was used as a stimulus. hK1 inhibitors VA999154 and VA999024 were added at a concentration of 0.5�� as described above. After 24h, the filters were mounted with DAPI to recognise the nuclei of migrated cells. Cell number was calculated by averaging the counts of five microscopic fields (photographed at �� 20). Endothelial cell migration assay The migratory response of ECs to GIST was assessed in a transwell array (Corning, Corning, NY, USA).
Briefly, 3 �� 105 GIST882 cells were plated on the bottom chamber of the migration system. Human umbilical vein ECs (HUVECs) were preincubated with B1R or B2R antagonists (Lys-des-Arg9Leu8-BK, LdL-BK or Icatibant, IC, respectively, 2 �� 10?7M) or PBS (vehicle), plated on top of the insert (50000 cell per insert) and then left to migrate overnight in the presence or absence of B1R or B2R antagonists. Migrated HUVECs were counted as described for the invasion assay and the percentage of migrated cells was calculated on the number of plated cells. Matrigel angiogenesis assay HUVECs pretreated for 30min with the hK1 inhibitor kallistatin (1��, R&D) (Wolf et al, 1999), the serine protease inhibitor Aprotinin (50Uml?1, Bayer, FRG) or vehicle were plated on growth factor-reduced Matrigel (BD Biosciences, Erembodegem, Belgium) in the presence of GIST882-conditioned medium or unconditioned medium (as a control).
Total tube length Batimastat and average tube thickness were measured on photographs captured at 24h and analysed using ImagePro Plus software (Media Cybernetics, Bethesda, MD, USA). FACS analysis Cells were incubated with rabbit polyclonal antibodies for B1R and B2R (1:100, Sigma), followed by FITC-labelled antirabbit secondary antibodies (Sigma) and analysed in a FACScalibur (BD Biosciences) flow cytometer. Controls were stained with isotype control.