Serial sections (3��m) were incubated overnight with monoclonal antibodies antihuman hK1 (1:100, R&D) and CD31 (1:40, DAKO) or Isolectin B4 (1:100, Sigma, St Louis, MO, USA) at 4��C. Immunoreactivity www.selleckchem.com/products/Bosutinib.html was detected using the ABC method (Vector Labs, Burlingame, CA, USA) or by applying a suitable fluorescence-labelled secondary antibody. Normal mouse IgG (Santa Cruz, Santa Cruz, CA, USA) was used as a negative control. Images were taken at �� 200 and �� 400 magnification using an Olympus light microscope (BX 40, Southall, UK). The stained area and intensity score were evaluated in the GNU Image Manipulation Program (GIMP) using colour selection and histogram function. HK1 staining intensity was recalculated to a 0�C100% scale, where zero represents no staining and 100 represents the darkest (black) area.
In CD31-stained samples, vessels were expressed as the average number of vessels per section. GIST cell line The immortalised GIST882 and GIST48 cell lines were a kind gift from Dr J Fletcher (Brigham and Women’s Hospital, Boston, MA, USA) (Tuveson et al, 2001). GIST882 was cultured in RPMI 1640 supplemented with 15% FBS, 2m -glutamine, 100IUml?1 penicillin and 100��gml?1 streptomycin (Cambrex, East Rutherford, NJ, USA). GIST48 was cultured in F10 (GIBCO) with 20% FBS, 2m -glutamine, 100IUml?1 penicillin and 100��gml?1 streptomycin, MITO serum extender and bovine pituitary extract (BD). Human umbilical vein endothelial cells (HUVEC, Cambrex) were grown in EGM-2 containing 2% FBS (Lonza, Basel, Switzerland). Conditioned medium Highly confluent GIST882 or GIST48 cells were incubated with serum-free medium for 24h.
After medium collection, cells were trypsinised and counted to establish total number. hK1 silencing Three to five million GIST882 cells were transfected with 150n of small interfering RNA (siRNA) for hK1 or scrambled control using Amaxa electroporation protocol (Amaxa Biosystems, Gaithersburg, MD, USA). Effective silencing was verified by RT�CPCR and ELISA (see below). On-target plus siRNA was purchased from Dharmacon (Lafayette, CO, USA). Infection with adenoviral vector carrying hK1 GIST882 cells were incubated with 50 moi (multiplicity of infection) of Ad.hK1 or Ad.Null overnight. Experiments were performed 24h later. Successful infection was verified by RT�CPCR (see below). Adenoviruses were prepared as previously described (Stone et al, 2009).
Measurement of hK1 levels and activity Immunoreactive hK1 in cell culture supernatants and blood was measured by ELISA as previously reported (Wang et al, 1995; Porcu et al, 2002, 2004) and hK1 activity was assayed using the chromogenic substrate S-2266, following the manufacturer’s instructions (Chromogenix, Milano, Italy) and as previously described (Madeddu Entinostat et al, 1993). Expression analysis RNA was obtained using an RNeasy Minikit (Qiagen, Crawley, UK) and reverse transcribed with MMLV (Invitrogen, Paisley, UK).