Porcine gelatin

Unless otherwise stated, coagulation factors were from Haematologic Technologies, Essex Junction, Vermont, USA. Porcine gelatin, bovine serum albumin (BSA) and conjugated antibodies were from Sigma-Aldrich, Suffolk, UK. Chromogenic substrates for ELISA were from KPL, Gaithersburg, Maryland, USA.

E14 mouse embryonic stem (mES) cells were cultured on 0.1% porcine gelatin (Sigma) coated 6-well plates at 37°C and 5% CO₂ in GMEM (Invitrogen) supplemented with 10% foetal bovine serum, 10³ U/ml leukaemia inhibitory factor (Millipore). Retinoic acid (Sigma) or DMSO (Sigma) vehicle was added to the media and refreshed every 24 hours.

Human embryonic kidney 293T and 293A cells (HEK-293T and HEK-293A, respectively; ATCC, Manassas, VA, USA) and human cervical adenocarcinoma cells expressing a tetracycline-inducible repressor (HeLa Tet-Off; Clontech, Mountain View, CA, USA) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine (Gibco). Pooled human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland) were cultured in endothelial cell growth medium 2 (EGM-2; Lonza). For HUVEC passaging, tissue culture plates were precoated for 30 min at 37°C with 0.1% (wt/vol) porcine gelatin (Sigma, St. Louis, Missouri, USA) in 1× phosphate-buffered saline (PBS; Gibco). All cells were routinely tested for mycoplasma by PCR method.

Confluent ovarian cancer cells were maintained in serum-free media for 24 h and the culture media was collected and analyzed using the gelatin zymography assay. Equal amounts of media were mixed with the SDS sample buffer without β-mercaptoethanol and loaded onto 10% SDS-PAGE gels containing 0.1% porcine gelatin (Sigma, USA). Electrophoresis was performed in an ice bath to prevent enzyme activation. Gels were then washed 4 times with gentle agitation for 15 min each with elution buffer (2.5% Triton X–100, 50 mmol/L Tris-HCL, 5 mmol/L CaCl2 and 1 μmol/L ZnCl2, pH 7.6) and 2 times for 20 min each with wash buffer (50 mmol/L Tris-HCL, 5 mmol/L CaCl2 and 1 μmol/L ZnCl2, pH 7.6). Gels were then placed in incubation buffer (50 mmol/L Tris-HCL, 5 mmol/CaCl2, 1 μmol/L ZnCl2 and 0.05% NaN3) and incubated at 37 °C for 48 h. Finally, gels were stained with staining buffer (0.05% Coomassie brilliant blue R-250, 30% methanol and 10% acetic acid) for 3 h and destained with destaining buffer (30% methanol and 10% acetic acid) for 3 h at room temperature. The bands were detected and quantified with Image Lab^TM Software.